The experiment aims to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from chicken and pig farms in Hunan province, China. Methods: A total of six tet(X4) positive strains were identified in 257 E. coli derived from chicken samples in Xiangtan city (n = 2), pig samples in Xiangxiang city (n = 1), Chenzhou city (n = 2), and Zhuzhou city (n = 1). The presence of tet(X4) was directly detected by PCR assay, and then the broth dilution method determined the antimicrobial susceptibility profile of the tet(X4)−positive isolates. Genomic locations were identified by whole−genome sequencing (WGS) and bioinformatics. Results: Almost all tet(X4)−positive strains showed high resistance to multidrug, including tigecycline. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to tetracyclines, β−lactams, phenicols, quinolones, lincosamides chloramphenicol, aminoglycosides and sulfamids. These tet(X4)−bearing strains exhibited six distract STs, such as ST10, 202, ST218, ST362, ST2077, ST7068. The plasmid replicon types carrying tet(X4) were the hybrid plasmid IncFIA(HI1)/IncHIA/IncHIB(R27) (5/6) and IncX1 (1/6). Conclusions: The presence of similar genetic environments in E. coli from different cities suggests there may be horizontal transmission pathways promoting the broad spread of drug−resistant genes in Hunan Province, putting great pressure on multidrug resistance monitoring.Background: The plasmid−mediated tigecycline resistance gene  HowevertetX/tetX4)  tet(X3)/from tetX [tetX6)tet(X3)/tX6) [tetX [tet(X13tetX14) , and tet samples ,12,13,14 , and te14) [tetX [tet(X4) tet(X4) . Mobile tet(X4)−positive E. coli isolates in chicken and pig farms in Hunan Province, China, to provide experimental data and a basis for drug resistance investigation and surveillance.Chickens and pigs are the main farmed animals in Hunan Province, with the emergence of multidrug resistance genes conferring resistance to most antibiotics in farms, bringing great pressure to daily management and limiting the use of antibiotics. In this study, we describe the characteristics and molecular epidemiology of tet(X4) gene were collected from four cities located far apart, with the prevalence rate of tet(X4) positivity at 2.33% (6/257). These tet(X4)−positive strains were from chicken fecal samples from Xiangtan city (2/80) and pig fecal samples from Chenzhou city (2/75), Xiangxiang city (1/52), and Zhuzhou city (1/50), with prevalence rates of 2.5%, 2.67%, 1.93%, and 2%, respectively (tet(X4)−positive strains are low compared to reports from chicken or pig farms in Jiangsu (18.24%) [tet(X3) was detected in food samples in Acinetobacter from 2015–2018 in Hunan province, China. The emergence of the tet(X4) gene was first reported in these cities and may be attributed to daily farm management and potential transmission.A total of six isolates positive for the ectively . This pr18.24%) , Shandon4% , Shantet(X4)−positive isolates showed resistance to multiple drugs; they were all resistant to ampicillin (≥256 mg/L), florfenicol (≥64 mg/L), gentamicin (≥64 mg/L), nalidixic (≥8 mg/L), trimethoprim–sulfamethoxazole (>16 mg/L), and tigecycline (≥4 mg/L), but susceptible to amikacin (0.5 mg/L), cefotaxime (≤0.125 mg/L), meropenem (≤0.5 mg/L), and colistin (≤0.25 mg/L) (blaTEM−1 (6/6), tet(X4) (6/6), floR (5/6), lnu(G) (5/6), and qnrS1 (5/6) were the most common ARGs in the six isolates. Strain 22a10, belonging to ST10, showed the most resistance genes. Interestingly, although the blaTEM−1 resistance gene existed in six strains, and the blaLAP−2 resistance gene existed in strain 22a62 in particular, these tet(X4)−positive strains did not show resistance to cefotaxime. Six tet(X4)−carrying strains exhibited high resistance to multiple classes of antibiotics, especially tigecycline, which could bring about great difficulty in clinical treatment.According to MICs (minimum inhibitory concentrations) compared with the resistance point in CLSI, these 25 mg/L) . It is n25 mg/L) . blaTEM−tet(X4)−positive strains were sequenced through whole−genome sequencing and used for phylogenetic tree analysis and other bioinformatics analyses. The phylogenetic tree analysis revealed that six tet(X4)−carrying isolates had genetic diversity (tet(X4) gene was extensive in various ST types. Importantly, one tet(X4)−positive E. coli, named 22a10, belonged to ST10, which was regarded as the main ST type for transferring tet(X4) [These iversity  and wereiversity . AlthougvirB) were found in almost all isolates, related to the transferability of bacteria and the contribution of tet(X4) to other bacteria [tet(X4) and blaTEM−1 resistance genes (tet(X4)−carrying plasmid replicon playing an important role in the spread of the tet(X4) resistance gene [tetX4 (MW940625), pSY3626 (CP059284), pT16R−1 (CP046717), and pE−T306 (CP090284) in E. coli SX8G, E. coli SY3626, E. coli T16R−1, and E. coli E−T306 derived from pork, chicken meat, pet dog, and human in China, respectively. In addition, plasmid p22a62 also showed 99% identity at 73% coverage to plasmid pHNGS471−2 (CP089511) in Klebsiella pneumoniae strain GD21SC417 isolated from Jiangsu Province in China (tet(X4)−bearing plasmid between animals and humans indicated that the plasmid had achieved a wide distribution among different origins, and the transmission of multidrug resistance, including tigecycline resistance, required more control and monitoring to reduce the potential risk of public food health problems [Through bioinformatics analysis, type IV secretion systems −harboring strains showed the same genetic environment compared with the structure ISCR2/tet(X4)/RdmC/hp/IS1R of the original tet(X4)−carrying plasmid pRT18−1 (MT219824)  [IS1 mobile elements have also been reported for the transmission of tet(X4) [tet(X4) by two accustomed IS elements calls for increased monitoring, and this identical group of genetic contexts that occurred in most farms may indicate that the structure ISCR2−tet(X4)−RdmC−hp−IS1R was conducive to the spread of tet(X4) [ISVsa3  . On the  tet(X4) . The tra tet(X4) ,23. Furt [ISVsa3 , which ctet(X4) from six strains were successfully transferred to E. coli C600, with transfer frequencies ranging from 10−6 to 10−4. This result indicated that the tet(X4) genes were located on a plasmid, and the hybrid−type replicons showed greater transfer compared with strain 22a22, which harbored IncX1−type replicons. Additionally, the horizontal dissemination of tet(X4) by conjugative plasmids or other mobilizable genetic elements existed in most chicken and pig farms in Hunan province, China, which may have accelerated the transmission of tet(X4).Plasmids carrying tet(X4) has been detected in farms in these cities in 2021, in addition to the prevalence of tet(X4)−positive E. coli at a low percentage compared with other provinces in China. Furthermore, these tet(X4)−harboring strains carrying similar genetic structures with extremely similar resistance phenotypes, plasmid types, and genetic environments were seen over a wide area. In addition, these tet(X4)−positive E. coli also had very high similarity to tet(X4)−positive strains detected in pets, farm animals, and humans in other provinces, piqued our research interest. According to what we know, the breeders in each farm are generally fixed and have no contact with each other, and the medications used in the farms are different. We hypothesize that (1) this plasmid type and gene structure are more prevalent in and conducive to the process of drug resistance transmission, and (2) the environment acts as a major reservoir of drug resistance genes, with some of these potential transmission pathways facilitating the transmission of drug resistance genes. However, this experiment suffers from insufficient sample data and requires further investigation and research.In summary, this is the first time that n = 80) and pig fecal samples in Chenzhou City (n = 75), Xiangxiang City (n = 52), and Chaling City (n = 50). All samples were kept in an icebox and transported to the laboratory. Then, the samples were cultured on MacConkey agar  and incubated at 37 °C overnight. A single pink clone was randomly selected [tet(X4) gene was identified in all isolates by PCR assay and Sanger sequencing using the primes described previously in A total of 257 samples were obtained in 2021 from chicken fecal samples in Xiangtan City (tet(X4)−positive isolates were subjected to antimicrobial susceptibility testing for 11 antimicrobial agents  using the broth dilution method and interpreted according to the American Clinical and Laboratory Standards [E. coli strain ATCC 25922 served as a quality control strain.The tandards . E. colitet(X4)−positive isolates using the TIANamp Bacteria DNA Kit DP302 . The whole−genome sequence of strains was determined using Illumina HiSeq 2500 . The draft genome sequences of six tet(X4)−positive E. coli were assembled by Spades 3.14 [https://patricbrc.org/, accessed on 8 August 2022). The sequence types and plasmid replicon types were analyzed using the CGE server (https://cge.cbs.dtu.dk/services/), and the phylogenetic trees were generated using Parsnp  and visualized using iTOL (https://itol.embl.de). Ultimately, to visualize the comparative genetic features, Easyfig v2.2.3 was used to generate linear comparison figures (http://mjsull.github.io/Easyfig).DNA was extracted from the des 3.14 . The asstet(X4) gene in the tet(X4)−positive strain was determined by conjugation experiment using E. coli C600 (streptomycin−resistant strain) as the recipient strain [Transferability of the t strain . The dontet(X4)−bearing strains from chicken and pork samples from various cities in Hunan Province, China. All tet(X4)−carrying strains exhibited high resistance to tigecycline and conferred resistance to multiple classes of antibiotics, which would bring about great difficulty in clinical treatment. Thus, we recommend using susceptible antibiotics to treat some bacterial infections in the investigated farms. Furthermore, the diversity of MLST types showed that tet(X4) genes have widespread sources in E. coli. Moreover, this study regarded the ISCR2−tet(X4)−RdmC−hp−IS1R structure as the dominant transmission potential pathway. Further attempts to reduce the risk of multidrug resistance transmission should focus on the mechanism mediated by mobile elements. In conclusion, the incidental transmission of multidrug resistance genes requires the rational use of antibiotics and improvements in strict daily management on the farm.In conclusion, we isolated six


We also assessed the cross-reactivity of the viruses with human monoclonal antibodies (mAbs) against H7N9 HA and ferret antisera against H7N9 AIV candidate vaccines. The H7N9 AIVs from the early epidemic waves had dual sialic acid receptor binding characteristics, whereas the more recent H7N9 AIVs completely lost or retained only weak human sialic acid receptor binding. Compared with the H7N9 AIVs from the first epidemic wave, the 2020/21 viruses formed larger plaques in Madin-Darby canine kidney (MDCK) cells and replicated to higher titres in ovo, demonstrating increased acid stability but reduced thermal stability. Further analysis showed that these recent H7N9 AIVs had poor cross-reactivity with the human mAbs and ferret antisera, highlighting the need to update the vaccine candidates. To conclude, the newly emerged H7N9 AIVs showed characteristics of typical AIVs, posing reduced zoonotic risk but a heightened threat for poultry.Since the first human case in 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human infections with a mortality rate of approximately 40%. Despite large-scale poultry vaccination regimes across China, the H7N9 AIVs continue to persist and evolve rapidly in poultry. Recently, several strains of H7N9 AIVs have been isolated and shown the ability to escape vaccine-induced immunity. To assess the zoonotic risk of the recent H7N9 AIV isolates, we rescued viruses with hemagglutinin (HA) and neuraminidase (NA) from these H7N9 AIVs and six internal segments from PR8 virus and characterized their receptor binding, pH of fusion, thermal stability, plaque morphology and  A new H7N9 virus strain emerged in China in 2013 and has resulted in more than 1500 confirmed human infections with ∼40% case fatality rate . In the Recently, Chen et al.  isolatedStudies investigating transmission of H5N1 AIVs in ferret model revealed that efficient human-to-human transmission requires shift from avian to human receptor binding preferences, reduced pH of fusion and increased HA thermal stability ,12. The In this study, we systematically investigated the risk of the newly emerged H7N9 AIVs to human and animal health by assessing the virus receptor binding, pH of fusion, HA thermal stability, virus infectivity and the cross-reactivity of these H7N9 variants with human H7N9 mAbs and ferret sera raised against World Health Organization (WHO) vaccine candidates.The receptor binding switch from avian-like to human-like is the key prerequisite for the interspecies transmission of AIVs from birds to humans. To examine such properties of the H7N9 AIVs in China, we utilized biolayer interferometry to characterize the receptor-binding profiles of these viruses to the avian-like 3′-sialylacetyllactosamine (3SLN) and human-like 6′-sialylacetyllactosamine (6SLN) receptor analogues. Three H7N9 AIVs isolated from December 2020 to March 2021 from three different provinces in China were selected for this study; they are HPAI H7N9 A/Chicken/Yunnan/1004/2021 (referred to as YN/21) from Yunnan province, south of China, HPAI H7N9 A/Chicken/Hebei/1009/2020 (referred to as HB/20) from Hebei Province, north of China, and HPAI H7N9 A/Chicken/Shanxi/1012/2021 (referred to as SX/21) from Shanxi province, north of China [The pH of fusion has been shown to play an important role in host adaptation and transmission; human-adapted influenza viruses generally fuse at a lower pH, while AIVs normally have a higher pH fusion threshold . Consistin ovo, indicating that these newly emerged H7N9 AIVs have a more balanced HA/NA function and possible more replication fitness than the viruses from the first epidemic wave.Efficient influenza virus replication requires an optimal balance between HA receptor-binding and NA receptor-destroying activities (reviewed in ). To assWe previously isolated and generated four monoclonal antibodies (mAbs) from humans naturally infected with H7N9 A/Anhui/1/2013 . The mAb2 reduction in HI titre with AH/13 ferret antiserum when compared with the AH/13  for influenza . To testhe AH/13 . Likewisin ovo. Moreover, these viruses also demonstrated reduced pH fusion thresholds, which has been shown to be essential airborne transmissibility for H9N2 and H5N1 AIVs between birds [To closely monitor the zoonotic risk of the newly emerged H7N9 AIVs, we selected three newly emerged H7N9 AIVs from different provinces of China from 2020 to 2021. Here we showed that the newly emerged H7N9 AIVs had typical AIV-like receptor binding profiles and reduced thermal stability, therefore posing a reduced zoonotic risk when compared to viruses from early epidemic waves. There was very low cross-reactivity of the newly emerged H7N9 AIVs with both mAbs isolated from humans naturally infected with H7N9 AH/13 and the ferret antisera against WHO candidate vaccines AH/13 and GD/16, highlighting the need to update the candidate vaccine strain. In addition to avian-like receptor binding profile, these newly emerged H7N9 AIVs showed increased replication fitness as evidenced by larger plaque sizes in MDCK cells and higher replication titres en birds ,27. TakiThe receptor binding specificity of influenza A virus is one of the major determinants of host specificity. It is a prerequisite for pandemic viruses to switch receptor binding specificity from avian receptor α2,3-SA to human receptor α2,6-SA . Here wein ovo growth (HA assay) showed a similar pattern as the plaque size. Interestingly, we noticed that GD/16 showed similar HA titre whereas displayed higher live virus particles detected by plaque assay when compared with AH/13. This difference might be linked to the receptor binding affinity as well. The allantoic fluid of GD/16 and AH/13 might contain similar number of virus particles, but due to the strong receptor binding affinity, GD/16 virus could enter the MDCK cells more efficiently than AH/13, therefore, a higher titre was detected with plaque assay. However, the strong receptor binding was not reflected in the HA assay due to its relatively low sensitivity when compared with the plaque assay. However, the precise mechanism remains to be elucidated.Efficient influenza virus replication requires an optimal balance between HA and NA (reviewed in ). HA iniin vitro and in ovo methods. Given the low risk to human infections, we believe the data here is of great importance for the policymakers to consider whether to keep the current vaccine scheme or adopt measures to eliminate the viruses from poultry. However, it is noteworthy that the work performed in this study comprised of in vitro and in ovo data with reassortant H7N9 AIVs with six internal gene segments from PR8 H1N1 influenza virus; in vivo experimentation and utilizing full wild-type viruses in the respective hosts would greatly strengthen the data generated in this study.Previously, we have shown that the A125T + A151T + L217Q mutations in HA could increase the viral HA stability when compared to AH/13. The A125T and A151T double mutations, which add glycan at amino acids 123 and 149, reduce the viral HA thermal stability whereas the L217Q significantly enhance the viral HA thermal stability . HoweverAll the procedures involving embryonated eggs were carried out in strict accordance with the guidance and regulations of United Kingdom Home Office regulations under project licence number P68D44CF4. As part of this process, the work has undergone scrutiny and approval by the animal welfare ethical review board at the Pirbright Institute. All the influenza virus-related work was carried out in biosafety level-2 conditions.The reassortant H7N9 viruses containing HA and NA from LPAI H7N9 (A/Anhui/1/2013) (referred to as Anhui/13), HA and NA from HPAI H7N9 virus (A/Guangdong/17SF003/2016) (referred to as GD/16), HA and NA from HPAI H7N9 virus (A/Chicken/Yunnan/1004/2021) (referred to as YN/21), HA and NA from HPAI H7N9 virus  or HA and NA from HPAI H7N9 virus . The DNA sequences for the HA and NA of H7N9 viruses are derived from the Global Initiative on Sharing All Influenza Data (GISAID). The GISAID accession numbers for the HA of Anhui/13, GD/16, YN/21, SX/21 and HB/20 are EPI439507, EPI439509, EPI1856859, EPI1857090 and EPI1857209, respectively. The GISAID accession numbers for the NA of Anhui/13, GD/16, YN/21, SX/21 and HB/20 are EPI919607, EPI1010185, EPI1856861, EPI1857092 and EPI1857211, respectively. The HAs and NAs were synthesized by GeneArt (Thermo Fisher Scientific) and then subcloned into PHW2000 plasmids. The polybasic amino acids motif of the HPAI H7N9 HAs were removed. The internal gene segments from PR8 H1N1 virus (A/Puerto Rico/8/34) were generated as previously described [2.The MDCK cells, human embryonic kidney 293 T and Vero cells (ATCC) were maintained with Dulbecco’s Modified Eagle’s medium (DMEM) (Gibco), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco) and 10% fetal calf serum (FCS) (Gibco), at 37°C with 5% COThe monoclonal antibodies L4A-14, K9B-122, L3A-44 and L4B-18 were generated by co-transfection with antibody light and heavy chain plasmids using ExpiFectamine™ CHO Transfection Kit according to the manufacturer’s protocol. The supernatants containing antibodies were filtered and purified by HiTrap Protein G HP antibody purification columns .Post-infection ferret antiserum raised against HPAI H7N9 virus GD/16 was kindly provided by Prof. Yoshihiro Kawaoka. The post-infection chicken antiserum raised against reassortant LPAI H7N9 AH/13 was generated by infection of specific-pathogen-free (SPF) Rhode Island Red chickens  with the virus . The serThe plaque assay was carried out as previously described , MDCK ceThree ten-day-old embryonated chicken eggs were infected with 100 plaque-forming units of H7N9 AIVs. The eggs were incubated at 35°C for 48 h before allantoic fluid was harvested and titrated by HA assay and plaque assay.The HA titration assays were performed with 1% chicken red blood cells as described in the WHO animal influenza training manual . Briefly25 μl of ferret sera or human mAbs were two-fold serially diluted in 96-well V-bottom plates (Greiner) with PBS prior to being mixed with equal volume of H7N9 Anhui/13 (8 HA units). The concentration for human mAb L4A-14, K9B-122, L3A-44, L4B-18 is 1.0, 1.5, 0.8, and 1.0 mg/ml respectively. The antibody-virus mixtures were incubated for 1 h at room temperature and then mixed with 50 μl of 1% chicken RBC. The plates were incubated for further 30 min at room temperature before the HI titre was recorded as the log2 of the reciprocal of the dilution.The reassortant H7N9 AIVs were diluted with allantoic fluid harvested from non-infected embryonated chicken eggs to 64 HA units/50 μl. The viruses were then heated at 50°C, 50.7°C, 51.9°C, 53.8°C, 56.1°C, 58.0°C, 59.2°C and 60°C using PCR thermal cycler (Biorad) for 30 min or left at 4°C as control before HA assay.The pH of fusion for H7N9 AIV was determined by syncytium formation assays as previously described . The virH7N9 AIVs were purified as previously described . Brieflyp values < 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software). One-way ANOVA and Tukey’s multiple comparison test was used to analyse the differences between different groups.


The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells).Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout.Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells.This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail. Human cytomegalovirus (HCMV) is a betaherpesvirus that causes lifelong infection with latency and is endemic in both developed and developing countries , 2. Infe+ T cells involved in the innate response to bacterial and viral pathogens. MAIT cells were initially labeled invariant due to their consistent expression of the T cell receptor (TCR) alpha chain Vα7.2-Jα33  cells are a subset of predominantly CD87.2-Jα33 –18, howe7.2-Jα33 , 19. MAI7.2-Jα33 , the fem7.2-Jα33  and brea7.2-Jα33 . MAIT ce7.2-Jα33  or by co7.2-Jα33 . MAIT ce7.2-Jα33 , 25–34 a7.2-Jα33 , 35–38.MR1 is a non-classical MHC I-like molecule whose function is known to include presentation of vitamin B-derived metabolites to activate MAIT cells , 39. DurHere we report modulation of surface and total MR1 protein by HCMV infection, and implicate the HCMV US9 gene product as contributing to this phenotype. We also demonstrate the ability of HCMV infected cells to inhibit bacteria-induced activation of primary MAIT cells in a MR1-dependent manner.Primary human foreskin fibroblasts (HFs) (ATCC), 293-TREX cells (invitrogen), HEK293Ts (ATCC), human telomerase immortalised (hTERT) HFs , 52 and were groThe bacterial artificial chromosomes (BAC) derived viruses based on the low passage HCMV strain Merlin, RCMV1111 (Merlin) and GFP expressing RCMV1158 (Merlin-GFP), were described previously . AD169-U2 of UV light using a CL-1000 Ultraviolet Crosslinker . Successful inactivation of virus was determined by a lack of development of CPE after incubation of UV-irradiated virus with fresh monolayers of HFs. For infection experiments, viable or UV-irradiated virus was applied to cultures for 90 minutes before being washed off; this point immediately after removal of the virus was taken as time zero.Ultraviolet (UV) irradiation of virus was performed by applying 720mJ/cmE.coli for use in Jurkat.MAIT and PBMC MAIT cell activation assays were grown by shaking at 37° overnight in LB broth then fixed in 1% formaldehyde for 3 mins, vortexing for the first 60s and last 30s :GCCCAGGCCGACGAGGAGGCGCAGCCACCGCCTCATGGCGGCTTCGCCAGAGCCGCCGGCACCGCGGCCGGCCGCAGGAAGCCGCCCGGCGCGTCGTCTGRecombinant adenoviruses (RAds) expressing C-terminally V5-tagged gpUS7 and gpUS9 were generated as described previously using recombineering technology . The codE.coli (1000 CFU/HF) was added in folate free RPMI 1640 supplemented with FCS and penicillin streptomycin. 4 hours later E.coli was removed by washing with PBS and LEAF-purified anti-MR1 neutralizing antibody  or appropriate isotype control was added for 1 hour (5 µg/mL) in folate free RPMI supplemented with FCS and penicillin streptomycin. Jurkat.MAIT cells were added at a Jurkat.MAIT : HF ratio of 2:1 in folate free RPMI supplemented with FCS and penicillin streptomycin. 20 hours later Jurkat.MAITs activation was assessed by flow cytometry.HFs were infected with HCMV in supplemented DMEM at MOI 10. 72 hours post infection (hpi) HFs were washed in PBS and partially fixed E.coli (1000 CFU/HF) was added to the infected HFs overnight. HFs were washed extensively in PBS to remove E.coli and if applicable LEAF-purified anti-MR1 neutralising antibody  or appropriate isotype control was added for 1 hour in folate free RPMI 1640 supplemented with human serum and penicillin streptomycin. PBMCs resuscitated the day before were added at a PBMC : HF ratio of 2:1 in folate free RMPI 1640 supplemented with human serum and penicillin streptomycin. After 5 hours of coculture PBMCs were stained for analysis by flow cytometry.HFs were infected with HCMV for 48 hours. Partially fixed Genome editing using CRISPR/Cas-9 was performed with the dual-vector lentivirus GeCKO system as described previously . BrieflyCell lysates from MR1 overexpressing cells were harvested in cell lysis buffer  as described previously . LysatesCells collected for analysis by flow cytometry were washed with PBS and stained for viability with Zombie NIR fixable dye (BioLegend). Surface staining followed with cells resuspended in FACS buffer (PBS supplemented with 1% FCS and 10 mM EDTA) and relevant antibodies added for 30 min at 4°C. Cells were acquired on a BD LSR II cytometer . Single stained UltraComp eBeads™ were washed in FACS buffer and fixed in the same way as surface stained cells, were used as compensation controls. FCS files were exported and analysed in FlowJo Software .® 700), CD69 . Matched isotype control antibodies were used where appropriate. Statistical analysis was performed in GraphPad Prism; when using the two-way ANOVA with multiple comparisions, Sidak’s correction was employed when the variance of both data sets needed to be taken into account e.g. neutralizing antibody and isotype control or knockout and parental cells. Tukey’s correction was used when comparisions were made within one set e.g. mock vs. mock + E.coli vs. HCMV vs. HCMV + E. coli within the neutralizing antibody treated cells only.The following antibodies were used to assess activation by flow cytometry: MR1 , MHC I , MICA , CD3 , CD161 , TCR Vα7.2 , CD8  downstream of the HCMV IE2 (Merlin-GFP) . GFP expFlow cytometry gates were established to separate GFP- bystander cells from GFP+ infected cells in cultures exposed to Merlin-GFP . This anTo determine if HCMV downregulated total cellular MR1 protein levels, HFs engineered to overexpress MR1  were mocTo identify potential HCMV proteins regulating MR1 protein expression, interacting proteins were investigated using a co-immunoprecipitation and mass spectrometry-based approach . HF cellThe ability of US9 to regulate MR1 surface expression was assayed using a replication deficient adenovirus (RAd) expressing US9. HFs transduced with RAd US9 or control Rad were treated with Ac-6-FP then stained for surface MR1 or MHC I , confirmvia an internal ribosomal entry site (IRES) upstream of the GFP open reading frame , intE.coli (a known driver of MAIT cell activation), described previously  for 48 hours before being exposed to partially fixed in Vα7.2 , 76, 77,in Vα7.2 , 78, 79,E. coli  to measure the degree of MAIT cell activation, there was no significant difference in the MAIT activating capacity of mock infected or HCMV infected primary HFs in the absence of To further examine the ability of HCMV to inhibit MR1-dependent MAIT cell activation, MR1 knockout fibroblasts were engineered using CRISPR/Cas-9 technology. The dual-vector lentivirus GeCKO system  was usedE. coli  as illustrated in This study builds upon our previous reports identifying that suppression of MR1 by viral infection is a feature of herpesvirus infection , 50. HergpUS9 is a glycoprotein that is a member of the US6 gene family of HCMV. A number of functional activities have been previously ascribed to gpUS9 including regulation of type I interferon responses by targeting MAVS and STING , regulatgpUS9 targets MICA*008 for proteasomal degradation with the N-terminal signal peptide recently implicated as playing a key role in the specific targeting of MICA*008 , 64. AltE. coli as an activating stimulus, HCMV was capable of efficiently inhibiting MAIT cell activation using both primary MAIT cells and reporter Jurkat.MAIT cells. To confirm that such regulation was dependent on MR1, two approaches were tested, namely genetic ablation of MR1 by CRISPR/Cas-9 technology as well as MR1 neutralization by specific antibodies. Both approaches identified that HCMV suppression of MAIT TCR dependent activation was MR1 dependent.The best characterized role of MAIT cells to date in responding to viral infection is through virus induced, cytokine mediated activation of MAIT cells, independent of the MR1:TCR axis , 78, 79.In the context of allogeneic hematopoietic stem cell transplantation (HSCT), where HCMV reactivation from latency is a frequent and often life-threatening complication in HSCT recipients, increased HCMV replication has been linked to development of acute and chronic graft vs. host disease (GVHD) , 85, andThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Sydney Human Research Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CA, BM, and RM performed experiments. CA, BM, HM, RM, AA, and BS designed experiments and/or analyzed data. CA, BM, AA, and BS wrote the manuscript. HM, RS, CF, DF, JM, JV, and JR provided reagents and/or key planning. All authors contributed to the article and approved the submitted version.


Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth. A subset of HIV-1 infected individuals generates antibodies that neutralize many different viral strains. However, this process requires years of infection and has proved difficult to achieve through vaccination. The envelope proteins of the infecting HIV-1 virus likely contain features that drive neutralization breadth within an individual. To explore this, we immunized rhesus macaques using envelope proteins from two HIV-1 infected human subjects that developed either high or low neutralizing antibody levels during infection. Several animals developed neutralizing antibodies when immunized with the envelope protein derived from the high neutralizing individual. Using viral escape variants that emerged in this infected individual over time, we mapped the targets of vaccine-elicited neutralizing antibodies to a distinct loop region of the envelope protein, which was also targeted by neutralizing monoclonal antibodies isolated from both the infected individual and the immunized rhesus macaques. Modeling of the envelope protein used in the vaccine demonstrated why this loop is vulnerable to antibody attack in infection and vaccination. By recapitulating the early HIV-1 human neutralizing antibody responses in a nonhuman primate immunization model, we demonstrate remarkable similarities as well as the potential of using this approach as a starting point to drive neutralizing antibody breadth, one of the ultimate goals of vaccine design. The HIV-1 envelope (Env) glycoproteins (gp) contain the determinants of entry into a host cell and are the targets of neutralizing antibodies (nAb). However, Env has heavy glycosylation that is extremely heterogeneous, particularly on the surface subunit gp120, resulting in a dynamic shield that often conceals conserved nAb epitopes . A recenIn a previous study, we ranked the level of plasma neutralization breadth present at 3 years after infection for 21 individuals in Zambia and Rwanda that were infected by subtype C, A, or AC recombinant HIV-1 viruses . ImportaHere, we demonstrate that all four vaccine regimens elicited high titers of antigen-specific IgG in serum that was broadly cross-reactive against diverse Env proteins. However, only two RM, both of which received the Z1800M T/F Env DNA/MVA-gp120 regimen, developed autologous nAb. For the two neutralization-positive RM, there was no evidence of serum neutralization breadth; however, the autologous nAb from both immunized RM was dependent on the gp120 V5 region. Neutralization was abrogated by a pair of amino acid substitutions or a single amino acid deletion in V5 that were found in early plasma nAb escape variants from Z1800M. These changes also conferred resistance to an early neutralizing mAb isolated from this subject. Profiling and modeling of glycan coverage on the Z1800M T/F Env gp120 immunogen supported that the V5 region is highly exposed on the T/F Env and that this inherent feature drove nAb targeting in subject Z1800M and in two immunized RM. Finally, an expanded family of V5-dependent mAbs was isolated from one of the neutralization-positive animals and these demonstrated identical neutralization characteristics to both the RM serum and the human Z1800M mAb 1A8. The results demonstrate that the Z1800M T/F Env nAb responses elicited in immunized nonhuman primates converge with those of the HIV-1 infected human subject from which the Env was isolated, highlighting the importance of Env immunogen selection and characterization, as well as the potential for using this as a platform to drive breadth.env genes were deposited into Genbank (KX983519.1 for R66M and KX983907.1 for Z1800M). The gp120 proteins used for immunization contained the wildtype sequences with a stop codon introduced into the Env open reading frame after HXB2 residue 511. Gp120 proteins were expressed and purified from 293F cells and antigenic characterization was conducted with reference bnAbs and non-neutralizing mAbs that recognize known epitopes through biolayer interferometry (BLI) [The wildtype T/F Envs, R66M_07Mar06_EnvO20 and Z1800M_26Jun07_EnvE4, are clade A/C recombinant and clade C, respectively, and have been described previously . The nucry (BLI)  and a biry (BLI)  2, making this protein cleavage independent [To generate the R66M and Z1800M trimers, we first evaluated whether each Env contained any of the 15 BG505-associated, trimer-derived (TD) stabilizing residues that were the basis for the native flexibly linked (NFL) approach used successfully to generate gp140 trimers for several diverse HIV-1 Envs . The R66ependent –26 . The Z1800M trimer contained a restructuring of a disordered region in gp41 HR1 (HXB2 547–569) to stabilize the prefusion conformation [4S)2 linker, and was truncated at HXB2 residue D664 . Therefore, we adopted the uncleaved prefusion-optimized (UFO) based approach described in ,28, whicormation , as wellormation , HXB2 Q1ormation , and HXBdue D664 . Size exdue D664 .The R66M NFL and Z1800M UFO trimers were further characterized by negative stain EM (NSEM), again using BG505 NFL as a prototype . The refWe also characterized the antigenicity of the R66M and Z1800M trimers using BAMA and BLI  Figs, asin vivo [We next tested whether vaccines based on the Z1800M and R66M T/F Envs could recapitulate features of nAb elicited in the human subjects from whom they were isolated. Stabilized Env trimer immunogens such as BG505 SOSIP have demonstrated the ability to elicit autologous nAb; however, these proteins do exhibit differences from the native Env, including altered glycosylation ,39 and tin vivo ,42. Furtin vivo . The co-The vaccines were administered to young adult male (n = 17) and female (n = 3) RM through intramuscular injection. The regimen, shown in Antigen specific serum IgG was measured by binding to the matching gp120 protein in ELISA and was evaluated at two weeks after each immunization . All fouWe next conducted a more extensive profiling of serum IgG using BAMA to evaluate recognition of globally diverse Envs. Antibody binding to a previously described panel consisting of 8 gp120, 8 gp140, and 16 V1V2 scaffold antigens representing clades A, B, CRF01_AE, CRF07_BC, and C from different geographic regions was measured, in addition to the 4 immunogen proteins . This pa50 titer could not be calculated with the least diluted serum concentration of 1:20  expressing the autologous T/F Env, Z1800M or R66M, using the TZM-bl assay. Serum samples collected at two weeks after the MVA and protein immunizations  and 13 weeks after the final immunization (week 74) were serially diluted and evaluated. For the R66M DNA/MVA/gp120, R66M DNA/MVA/trimer, and Z1800M DNA/MVA/trimer vaccination groups, nAb was low to undetectable at all time points tested, and an ID of 1:20 . In contectively , and it ectively . The ID5We next tested whether IgG purified from the serum of ROa17, RLk17, and another RM from the same vaccine group that did not develop serum autologous nAb activity, RPz16, could mediate the observed neutralization. Using concentrations of purified IgG that were normalized to the IgG concentration of the serum, ROa17 and RLk17 nAb activity at weeks 55 and 63 was recapitulated Figs  and S6. Focusing on the two RM that developed autologous nAb against the Z1800M T/F Env, we tested two Envs, Z1800M_30Nov07_D10  and Z1800M_30Nov07_D11  that were previously isolated from human subject Z1800M at 5 months after infection . These EWe also tested the CD4bs bnAb VRC01 against the chimera and mutant panel. While this bnAb neutralized all constructs, it inhibited D10, D10_TF3, and D10_V5 more potently that the T/F Env, indicating that the changes in V5 increased exposure of the VRC01 epitope within the CD4bs . In contIn addition to longitudinal Envs isolated at 5-months from human subject Z1800M, mAbs were previously isolated at 7-months from B cells that were single cell sorted using the T/F Env gp120 as a probe . Of 64 m50 of 0.6 μg/ml but was unable neutralize Env D10 and D11 PV at 10 μg/ml  (5GlcNAc2 (Man5). These findings are consistent with previous studies that have demonstrated heterogeneity with a high proportion of complex type glycans on recombinant gp120 molecules [Vaccine elicited autologous nAb often target glycan hole regions that are exposed on both the immunogen and the native Env trimer ,13,40. BGlcNAc2) . The majolecules –51.The heterogeneity and flexibility of glycans over Env makes high resolution structural characterization of the glycan shield extremely challenging. We therefore employed computational methods to model the Z1800M Env glycoprotein and its glycan shield topology in atomistic detail as described ,53. The There are 6 N-linked glycans located proximally to the V5 loop on the T/F Env on glycosites N228, N270, N331, N347, N383, and N450 . Of these, the glycan at N450 (HXB2 N463) is spatially closest to V5 but is oriented such that it is inserted below the loop. This positioning of the glycan at N450 (HXB2 N463) in relation to V5 effectively results in a hole that exposes the V5 loop . As showTo further examine how a substitution in the protein backbone could have such a significant effect on the glycan N450 coverage of the V5 loop, we considered the V5 loop conformation as well. A detailed structural characterization of the V5 loop demonstrated that the positional deviation of the loop and the residue-wise fluctuations arising from the simulated annealing based structural relaxation of the models, are significantly higher in D10 and D11 compared to the T/F Env, especially on the C-terminal region of the loop . There aThe differences in glycosylation, steric as well as in number of glycosites, between the T/F Env and D10/D11 caused the glycan shielding to change over several regions on the modeled SOSIP surface . The priTo gain a higher resolution view of the nAb elicited by Z1800M DNA/MVA/gp120 immunization, antigen-specific IgG+ B cells from neutralization-positive RM RLk17 were sorted into single wells from week 63 cryopreserved PBMC, which was 2 weeks post-second gp120 immunization and the peak serum nAb time point, using previously described approaches . Using t50 titers ranging from 0.09 μg/ml to 0.68 μg/ml)  Figs  and 14A ml) Figs . The CDRml) Figs . FurtherFinally, we performed a competition ELISA to further define the epitope region on the T/F Env gp120 that is recognized by the nmAb clonotype. We selected 3 representative nmAbs for these studies. First, we tested whether pre-incubation with each of the 3 clonotype nmAbs reduced binding of the human nmAb 1A8 to the T/F Env gp120 protein . All 3 nThe premise underlying this study was that HIV-1 T/F Envs have inherent features that drive nAb responses during infection, and that those can be translated into vaccines to direct nAb at a certain target. In our case, we selected T/F Envs isolated from two HIV-1 infected individuals that showed distinct outcomes in terms of autologous and heterologous nAb development. We also delivered the Envs in multiple forms, as it remains unclear what the best immunogens are for eliciting autologous tier 2 nAb and for ultimately developing breadth. The findings demonstrated that the Z1800M T/F Env from the high nAb responder has an exposed, immunogenic V5 loop that was targeted by nAb in early infection and in vaccination, but only in the DNA/MVA/gp120 group. The Z1800M T/F Env DNA/MVA/trimer immunization regimen did not elicit nAb in any of the 5 RM, which could have been due to the heterogeneity of the trimer we generated. However, according to our modeling, V5 would have likely been exposed on Z1800M Env forms if it had taken on a stable trimer conformation. It could also be that the stabilizing change introduced near V5 HXB2 A433P) altered the exposure or conformation of this region. In contrast, none of the RM immunized with R66M T/F Env vaccines (from our low nAb responder) containing gp120 or trimer developed nAb. It could be that the R66M T/F Env does not have a region that is well exposed on the native trimer, as autologous nAb titers remained low to undetectable during HIV-1 infection 33P alter,56.A unique aspect of our study was the availability of acute infection and longitudinal samples from participants in Zambia and Rwanda . This enin vivo escape. This contrasts with serum nAb mapping studies of BG505 SOSIP immunized RM, where closing glycan holes results in only partial resistance [in vivo that could be counterproductive. A powerful way to use the Z1800M T/F Env could be to design immunogens that successively increase coverage of V5 and exposure of the CD4bs. Such re-direction of nAb could be achieved by altering the structural flexibility of the V5 loop or restricting the N450 (HXB2 N463) glycan in an orientation away from CD4bs. Moreover, CD4bs glycan coverage is relatively lower in Z1800M than other common Envs such as BG505 SOSIP  at Emory University and complied with NIH guidelines. Animal research was also in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to experiments involving animals. All animal research adhered to the principles stated in the 2011 Guide for the Care and Use of Laboratory Animals prepared by the National Research Council. Yerkes National Primate Research Center (YNPRC) is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Methods of euthanasia were consistent with the American Veterinary Medical Association with Guidelines.This study also utilized samples obtained previously from individuals enrolled in heterosexual discordant couple cohorts in Rwanda and Zambia ,71,72. AMacaca mulatta), including 17 male and 3 female, were included in this study. All animals were between 3 and 5 years of age at the start of the study. Immunizations were as follows: week 0 (lt. and rt. outer thigh) and week 8 (lt. and rt. outer thigh) with pGA1-SHIV-R66M/Z1800M DNA ; week 16 (lt. and rt. outer thigh) and week 24 (lt. and rt. deltoid) with rMVA-SHIV-R66M/Z1800M ; weeks 53 (lt. and rt. deltoid) and 61 (lt. and rt. deltoid) with either gp120 or trimer protein . All immunizations were performed intramuscularly with each immunization dose split equally between left and right anatomic sites. The RM were split into 2 groups of 10 animals each and one group received reagents derived from the HIV T/F Env Z1800M E4.02 sequence while the other received reagents derived from the HIVT/F Env R66M O20.03 sequence [20 Indian origin rhesus macaques , processed according to the manufacturer’s recommendations and purified PBMC were cryopreserved in 90% FBS/10% DMSO in liquid nitrogen vapor phase storage.gag to inactivate RNA packaging and into pol corresponding to protease and reverse transcriptase to inactivate their functions. All genes were expressed from a single promoter using the sub-genomic splicing that is used by HIV/SIV. Using a pGA1 DNA construct containing the HIV-1 C.1086 K160N Env as a base plasmid, the gp160 coding region was replaced with the corresponding open reading frames from R66M and Z1800M T/F Envs. The plasmid pGA1-SHIV-R66M was synthesized by GenScript and pGA1-SHIV-Z1800M was synthesized by Aldevron. The expression of Gag and the cognate Env was confirmed by flow cytometry (The DNA vaccines were constructed in a plasmid vector backbone of pGA1 consisting of a eukaryotic expression cassette with a CMV immediate early promoter (CMV-IE) and intron A that facilitates gene splicing, as described previously ,42,73,74ytometry . NucleotA wildtype MVA (isolated before 1974) was used to generate the recombinant MVA (rMVA) vaccines, as described previously ,42,74. TGalanthus nivalis lectin coupled agarose beads  reconstituted in an Econo-Column chromatography column  for purification of the protein. After overnight drip at 4°C, the column was washed with 1X PBS, and protein was eluted in 7–10 volumes of 1 M α-methyl mannopyrannoside and fractions were pooled. Purified protein was concentrated, and buffer exchanged into 1X PBS, 500 mM NaCl using an Amicon Ultra-15 centrifugal filter unit . The protein was further purified using Size Exclusion Chromatography using a HiLoad Superdex S200PG column  on an AKTA Pure HPLC system (GE Life Sciences). Peak fractions corresponding to monomeric gp120 were further visualized on Blue Native gel electrophoresis. The gp120 containing fractions were pooled and the final yield of each purified gp120 protein was ~1.5mg. Protein was stored in aliquots of 50 μl at a concentration of 2–3 μg/μl at -80°C.To produce the gp120 proteins, the nucleotide region 6225 to 7757 encoding amino acid residues 1 to 511 (HXB2 numbering) followed by a stop codon was synthesized for each T/F Env by GeneArt  using gene optimization and then subcloned into the pcDNA3.4 expression vector. Plasmids were transiently transfected into Expi293F cells using the ExpiFectamine 293 Transfection Kit  according to the manufacturer’s instructions. The transfected culture supernatant was collected at days 5–7, clarified via low-speed centrifugation, and passed through a 0.22 μm Steritop vacuum filtration unit . The clarified, filtered supernatant was passed through 2 and 80% humidity in a shaking platform incubator (Infors HT Multitron) at 120 rpm in baffled, vented flasks (VWR). After 5 days, supernatant was collected, with all subsequent procedures performed at 4°C. The supernatant was centrifuged at 3,500 rpm for 45 min, followed by filtration through a 0.22 μm Steritop vacuum filter . The clarified, filtered supernatant was passed through Galanthus nivalis lectin coupled agarose beads  reconstituted in an Econo-Column chromatography column  for purification of the protein. After overnight drip at 4°C, the column was washed with 1X PBS, and protein was eluted in 7–10 volumes of 1 M α-methyl mannopyrannoside and fractions were pooled. Purified protein was concentrated, and buffer exchanged into 1X PBS, 500 mM NaCl using an Amicon Ultra-15 centrifugal filter unit . SEC was performed using a 16/600 Superdex 200 Column  on an AKTA Pure HPLC system (GE Life Sciences). The BG505 NFL trimer DNA plasmid, kindly provided by Dr. Richard Wyatt, was used to express the BG505 NFL trimer as a procedural control. Trimeric protein containing fractions were collected, pooled, and concentrated in 1X PBS, 150mM NaCl. Trimer protein was aliquoted and stored at -80°C.To produce trimeric gp140 proteins, the nucleotide region 6225–8216 encoding amino acid residues 1 to 664 (HXB2 numbering) followed by two stop codons was synthesized for each T/F Env by GeneArt  using gene optimization and then subcloned into the pcDNA3.4 expression vector, with the following modifications: residues 1–29 were replaced with the CD5 leader sequence (MPMGSLQPLATLYLLGMLVASVLA) and residues 508–511 representing the furin cleavage site were replaced with a linker (GGGGSGGGGS). In addition, each construct contained a series of amino acid substitutions that had been described previously to stabilize other HIV-1 Env gp140 proteins. The R66M NFL contained V65K, A73C, Q106T, I201C, N302Y, E429R, R432Q, A433C, K500R, F519R, I520R, Q543N, I559P, T569G, K588R, A561C –21. The th percentile of all baseline sample binding levels per antigen, MFI > 3-fold of that of the matched baseline before and after Blank/MuLVgp70_His6 subtraction. BAMA was also used to measure HIVIG and monoclonal antibody binding to the immunogen proteins Z1800M gp120, Z1800M trimer, R66M gp120, R66M trimer. The immunogen proteins were directly coupled to polystyrene beads and binding of antibodies was measured using mouse anti-human IgG-biotin followed by streptavidin-PE. Each antibody-protein combination was tested in duplicate. PGT145 and PGT151 were tested starting at a concentration of 20 μg/ml followed by ten 4-fold serial dilutions. All other mAbs were tested at a single concentration of 20 μg/ml. HIVIG was tested starting at a 1:100 dilution followed by ten 6-fold serial dilutions  with the function lmer under the package lme4 1.1.26 in R 4.0.3. Time points and subject ID (antiID) were considered to have random effects, for which a random intercept as well as a random slope were added to the model. Four dummy variables were created and corresponded to four vaccines, respectively. Another dummy variable was created to indicate the type of protein (gp120 or trimer). The antigen gp70-TT31P.2F10.2792 V1V2 was excluded since data was missing and subject 6S0 was also excluded since the visits were not complete. The effects of the four vaccines were evaluated by LMM with time points (visits), specific vaccine and their interactions as the covariates. Additionally, the effects of the two proteins were compared with visits, proteins, and their interactions as the covariates. In the LMM model, the protein “gp120” served as the reference level.mAbs were recovered from single, antigen-specific B cells using procedures described previously for both HIV-1 infected subject Z1800M and immunized RM RLk17 ,15. The Immunoglobulin heavy and light chain variable domain regions were PCR-amplified as previously described, using oligo-dT  and SuperScriptIII  ,15,75. FBinding was assessed on an Octet RED96  at 30°C, with 1,000 rpm agitation. Each mAb was immobilized on an Anti-hIgG Fc Capture (AHC) biosensor  at a concentration of 25 or 10 μg/ml for 300 s, and a baseline reading was recorded for 60 s in kinetics buffer . Sensors were then immersed in varying molarities of the corresponding autologous T/F gp120 protein (0.1875 to 1.5 μM) or trimer (0.037 to 1 μM) for 300 s, then allowed to dissociate in kinetics buffer for 600 s. Sensors were regenerated before each kinetics assay. Human IgG at 25 or 10 μg/ml dipped into kinetics buffer was included on every assay plate for reference during analysis . Data was analyzed using ForteBio Data Analysis 9.0. Binning (competition) assays were performed at 30°C, with 1,000 rpm agitation. Synthesized Z1800M T/F gp120 at a concentration of 25 μg/ml was immobilized on an Anti-Penta-HIS  biosensor for 300 s, and a baseline reading was recorded for 30 s in kinetics buffer. Sensors were then immersed in 0.66 μM primary mAb for 10 minutes. After a second baseline reading for 30 s in kinetics buffer, sensors were immersed in 0.66 μM secondary antibody to assess reference mAb binding capacity in the presence of primary mAb binding . Reference mAb as primary and secondary mAb was included as a positive control for competition (reduced reference binding). Binning values were calculated using ForteBio Data Analysis 9.0.The VRC01 mAb VH and VL expression plasmids were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: from Dr. John Mascola (12035 and 12036) ,81; mAb 50 or IC50 titer values were calculated in Graphpad Prism using the dose–response inhibition analysis function with variable slope, log-transformed x values, and normalized y values.Neutralization was measured using serially diluted, heat-inactivated immunized RM serum, RM or human plasma, or mAbs in the TZM-bl assay as previously described, using cells plated one day prior to the assay ,58,83–942CO3 + 2.9g NaHCO3 in 1L H2O). The plates were washed 3x with PBS-T (0.05% Tween in PBS) and incubated for 2 hours in blocking buffer  followed by an additional 3 washes in PBS-T prior to addition of samples. Heat inactivated RM serum samples were diluted in blocking buffer to starting concentrations between 1:100 and 1:1000. This initial dilution was then serially diluted 3-fold in blocking buffer and 100 μl was added to each well (8 concentrations tested for each sample) and incubated for 2 hours at room temperature. After 6 washes in PBS-T, HRP-conjugated anti-monkey IgG  was added to each well at a 1:10,000 dilution in blocking buffer and incubated for 1 hour at room temperature. The plates were washed 6 times in PBS-T, and then tetramethylbenzidine SureBlue 1-component microwell peroxidase substrate  was added. The plate was incubated in the dark at room temperature for 15 min, at which time 4 N H2SO4 stop solution  was added. The optical density at 450 nm was determined on an Epoch Microplate Spectrophotometer (BioTek), and the data were analyzed using Gen5 software. Quantitation of gp120-specific serum IgG was performed by including a standard curve for IgG on each plate. For this, 2 columns of each plate were treated as above except for coating with goat anti-rhesus IgG unlabeled  and incubating with serial dilutions of Rhesus IgG . Concentrations of serum IgG that bound to gp120 were determined using all dilutions that fell within the quantitative range of the standard curve and were represented by the average for each sample. Each assay was run with duplicate wells and one independent repeat.To measure Z1800M or R66M-specific IgG antibodies in RM serum, 96-well plates  were coated at 4°C overnight with 1ng/μl Z1800M or R66M gp120 (produced as described above) in carbonate-bicarbonate buffer (1.5g Na2CO3 + 2.9g NaHCO3 in 1L H2O). The plates were washed 3x with PBS-T (0.05% Tween in PBS) and incubated for 2 hours with blocking buffer  followed by an additional 3 washes in PBS-T prior to addition of samples. An initial 5 μg/ml mAb were serially diluted 4-fold in blocking buffer and 100 μl was added to each well (8 concentrations tested for each sample) and incubated for 2 hours at room temperature. After 6 washes in PBS-T, HRP-conjugated anti-human IgG  was added to each well at a 1:10,000 dilution in blocking buffer and incubated for 1 hour at room temperature. The plates were washed 6 times in PBS-T, and then tetramethylbenzidine SureBlue 1-component microwell peroxidase substrate  was added. The plate was incubated in the dark at room temperature for 10 min, at which time 4 N H2SO4 stop solution  was added. The optical density at 450 nm was determined on an Epoch Microplate Spectrophotometer (BioTek), and the data were analyzed using Gen5 software. Each assay was run with duplicate wells and one independent repeat. For Z1800M T/F gp120 competition ELISAs, mAb b6, VRC01, Z1800M 1A8 and PGT121 were biotinylated using the EZ-Link-Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher Scientific) and ELISA assays were performed as above with minimal modification. Specifically, His-tagged Z1800M T/F gp120 protein was pre-bound to Qiagen Ni-NTA HiSorb 96 well plates (Qiagen 35061) for two hours. Following the 2 hour mAb incubation step performed as above  the plates were washed and incubated with biotinylated mAb for an additional 2 hours. This was followed by an additional 6 washes with PBS-T before the addition of 100 μl/well of a 1:200 dilution of Pierce High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific 21134) for 1 hour.To measure mAb binding to Z1800M Env protein, 96-well plates  were coated at 4°C overnight with 1 ng/μl Z1800M-T/F-His or Z1800M-V5-D11-His gp120 in carbonate-bicarbonate buffer (1.5g Na2SO4 stop solution (Fisher Scientific SA818-1) was added. The optical density at 450 nm was determined on an Epoch Microplate Spectrophotometer (BioTek), and the data were analyzed using Gen5 software. Quantitation of gp120-specific serum IgG was performed by including a standard curve for IgG on each plate. For this, 2 columns of each plate were incubated with serial dilutions of Rhesus IgG . Concentrations of IgG present were determined using all dilutions that fell within the range of the standard curve and were represented by the average for each sample. Each assay was run with duplicate wells.IgG was purified from 100 μl RM serum samples using protein G Ab Spin Trap columns (Cytiva LifeSciences) according to the manufacturer’s suggested procedure with a final elution volume of 200 μl. The amount of IgG present in the initial serum sample as well as in the flow through and elution fractions were quantified by ELISA. 96-well plates  were coated at 4°C overnight with 1 ng/μl Goat anti-rhesus IgG UNLB (Southern Biotech 6200–01) in carbonate-bicarbonate buffer. The plates were washed 3x with PBS-T (0.05% Tween in PBS) and blocked for 2 hours with blocking buffer  followed by an additional 3 washes in PBS-T prior to addition of samples. Heat inactivated RM serum samples, flow through and elution fractions from the column were diluted in blocking buffer to starting concentrations of 1:500. This initial dilution was then serially diluted 4-fold in blocking buffer and 100 μl added to the wells (8 concentrations tested for each sample) and incubated for 2 hours at room temperature. After 6 washes in PBS-T, HRP-conjugated anti-monkey IgG (Alpha Diagnostic Intl 70021) was added to each well at a 1:10,000 dilution in blocking buffer and incubated for 1 hour at room temperature. The plates were washed 6 times in PBS-T, and then tetramethylbenzidine SureBlue 1-component microwell peroxidase substrate (KPL 52-00-03) was added. The plate was incubated in the dark at room temperature for 15 min, at which time 4 N HCloning of HIV-1 Env expression plasmids R66M_07MAR06_O20, Z1800M_26JUN07_E4, Z1800M_30NOV07_D10 and Z1800M_30NOV07_D11 was described previously [Glycoproteomic sample preparation, nano-LC-MS/MS data acquisition and data analysis of monomeric Env gp120 Z1800M: Sequencing-grade modified trypsin and chymotrypsin were purchased from Promega . Sequencing-grade modified endoproteinase Glu-C was acquired from Roche Diagnostics . All other reagents were purchased from Sigma Aldrich unless indicated otherwise. Data analysis was performed using Byonic 2.3 software and manually using Xcalibur 4.2 and GlycoWorkbench 1.1. Protease digestion for glycoproteomics of gp120 Z1800M: The purified and lyophilized monomeric T/F Env gp120 protein Z1800M was dissolved in 50 mM ammonium bicarbonate solution. The protein was reduced in 12 mM dithiothreitol solution, and subsequently alkylated in 30 mM iodoacetamide. In total, three enzymatic digestions were set up for best coverage: (i) tryptic digest for 16 h at 37°C, (ii) tryptic digest (16 hours at 37°C) followed by a chymotryptic digest at room temperature for 16 hours, (iii) tryptic digest (16 hours at 37°C) followed by a digest with endoproteinase Glu-C at 37°C for 16 hours in phosphate buffer. The three digests were individually filtered through 0.2 μm filter and directly analyzed by LC-MS/MS. Data acquisition of protein digests using nano-LC-MS/MS: The glycoprotein digests were analyzed on an Orbitrap Fusion Tribrid mass spectrometer equipped with a nanospray ion source and connected to a Dionex Ultimate 3000 RSLC nano system . A pre-packed nano-LC column  of 15 cm length with 75 μm internal diameter (id), filled with 3 μm C18 material (reverse phase) was used for the chromatographic separation of the samples. The precursor ion scan was acquired at 120,000 resolutions in the Orbitrap analyzer and precursors at a time frame of 3 s were selected for subsequent MS/MS fragmentation in the Orbitrap analyzer at 15,000 resolution. The LC-MS/MS runs of each digest were conducted for 72 min. 0.1% formic acid and 80% acetonitrile-0.1% formic acid was used as mobile phase A and B, respectively, to separate the glycopeptides. The intensity threshold for triggering an MS/MS event was set to 2000 counts, and monoisotopic precursor selection was enabled. MS/MS fragmentation was conducted with a stepped HCD  product triggered CID (Collision-Induced Dissociation) (HCDpdCID) program. Charge state screening was enabled, and precursors with unknown charge state or a charge state of +1 were excluded (positive ion mode). Dynamic exclusion was enabled for an exclusion duration of 30 s after 2x detection within 20 s. Data analysis of glycoproteins: The LC-MS/MS spectra of all three enzymatic digests of the monomeric gp120 Z1800M mutant were searched against the FASTA sequence using the Byonic software 2.3 by choosing appropriate peptide cleavage sites (non-specific cleavage option enabled). Carbamidomethylation of cysteine was set as a fixed modification, whereby oxidation of methionine and common human N-glycans found in plasma (variable) were set as variable modifications. The LC-MS/MS spectra were also analyzed manually for the glycopeptides with the support of the Thermo Fisher Scientific Xcalibur 4.2 software, GlycoWorkbench 1.1., GlycoMod tool, and ProteinProspector v6.2.1. The HCDpdCID MS2 spectra of glycopeptides were evaluated for the glycan neutral loss pattern, oxonium ions and glycopeptide fragmentations to assign the sequence and the presence of glycans in the glycopeptides. Spectra for the most common glycoform at each site used for computer modelling are included in BioRender.com.Glycoprotein conformational ensemble was generated by a previously established high throughput atomistic modeling pipeline (HTAM),53. In t-/Å2, and a magnification resulting in a pixel size of 1.011 Å for BG505 and 1.648 Å for R66M and Z1800M. Images were acquired on a Gatan OneView CMOS camera with a nominal defocus range of 0.5 to 1 μm using the SerialEM program [EM analysis of R66M, BG505, and Z1800M Env trimers were performed as described before . Briefly program . Particl program ,104. Ext program . ParticlS1 Fig(A), the Z1800M UFO trimer and R66M NFL trimer (B), and the BG505 NFL trimer as a process control (C) were analyzed by measuring the binding of HIV bnAbs, non-neutralizing mAbs, and HIVIG through BAMA or BLI. For the BLI experiments, which were conducted on an Octet Red96, each antibody was immobilized on a biosensor at a concentration of 10 μg/ml (A) or 25 μg/ml (B) and the sensors were immersed in varying molarities of each protein. The gp120 proteins were tested at concentrations ranging from 0.1875 to 1.5 μM and the trimers were tested at concentrations ranging from 0.037 to 1 μM. The individual sensorgrams are shown in A-C, the antibody, binding specificity, and detection method is shown for each antibody-protein combination. The color intensity and number of plus signs indicate the relative strength of binding; gray boxes with a dash indicate that binding was below detection; NT indicates ‘not tested’. Some antibody-protein combinations  were analyzed using both methods and the results of both are shown.The Z1800M and R66M T/F Env gp120 proteins (TIF)Click here for additional data file.S2 Fig(A) The R66M and Z1800M gp120 immunogen proteins were analyzed by BLI, using an Octet Red96, using HIV bnAbs PG16, VRC01, PGT121, as well as non-neutralizing CD4 binding site mAb F105, and autologous non-neutralizing R66M mAb7A2 and autologous neutralizing Z1800M mAb 1A8 as controls. Each antibody was immobilized on a biosensor at a concentration of 10 μg/ml and the sensors were immersed in varying molarities of each protein. The gp120 proteins were tested at concentrations ranging from 0.1875 to 1.5 μM. (B) The R66M NFL trimer and Z1800M UFO trimer were also analyzed by BLI, using V3-glycan bnAbs PGT121, PGT128; V1V2 apex bnAbs PGT145, PGDM1400, PG9, PG16; CD4 binding site bnAb VRC06b and non-neutralizing mAb F105; CD4-induced non-neutralizing mAb 17b; and V3 bnAb 447-52D. The trimers were tested at concentrations ranging from 0.037 to 1 μM with antibody immobilized at 25 μg/ml. Response units are indicated on the y axis and are plotted against time on the x axis. Related to (TIF)Click here for additional data file.S3 FigThe fluorescence intensity data generated via antibody binding to the immunogen proteins in the BAMA assay is shown. Proteins were analyzed using V1V2 apex bnAb PGT145, gp120-gp41 interface bnAb PGT151, both with serial dilutions starting at 20 μg/ml; a panel of bnAb and non-neutralizing antibodies PGT125, 19B, 17B, VRC01, CH102, F105, 7B2, and an anti-influenza bnAb CH65 at 20 μg/ml; HIVIG at serial dilutions starting at 1:100; and normal human serum (NHS-60) diluted at 1:500 as a negative control. Related to (TIF)Click here for additional data file.S4 Fig(A) A schematic representation of the recombinant DNA plasmid that expresses SIVmac239 Gag/Pro/Pol and HIV-1 Env is shown. (B) Flow cytometric detection of HIV-1 Env (bnAb PGT121) and SIV Gag (mAb 2F12) expression in R66M and Z1800M T/F Env recombinant DNA plasmid transfected 293T cells. (C) Flow cytometric detection of SIVmac239 Gag and HIV-1 Env in DF-1 cells infected with the R66M and Z1800M T/F Env recombinant MVA vectors. Surface Env was stained using PGT121 and intracellular Env was detected using mAb ID6. Surface and intracellular Gag was detected using mAb 2F12. Gag and Env expression were plotted on live cells infected with rMVA, stained intracellularly for MVA E3 protein . (D) Lysates (L) and supernatants (S) from rMVA infected DF-1 cells were probed with serum from a BG505 SOSIP-immunized RM to detect HIV-1 Env on western blot and SIV Gag was detected using mAb 2F12.(TIF)Click here for additional data file.S5 Fig(A) and the subtype of the Env proteins in (B). The top row contains Z1800M T/F Env immunized RMs that received the trimer or gp120 immunogen, as indicated, and each line represents the mean response of RM in that group against one gp120/gp140/V1V2 binding antigen. The bottom row contains the same information for R66M T/F Env immunized RMs. The mean MFI (mean fluorescence intensity) was calculated for RMs in the four vaccination groups and is plotted on the y-axis on a log10 scale for each binding antigen. The time points of the immunizations are indicated on each graph. Error bars represent the standard deviation of mean serum IgG binding to the antigen panel for the RMs in each vaccination group.Serum IgG binding to 8 gp120 proteins, 8 gp140 proteins, and 16 scaffolded V1V2 antigens from the BAMA antigen panel, as well as the R66M gp120 and trimer and Z1800M gp120 and trimer, was analyzed with respect to time (weeks). The data was colored according to the type of protein in (TIF)Click here for additional data file.S6 FigIgG was purified from serum at week 55 and 63 from RLk17 and ROa17 and tested for neutralization of the Z1800M T/F Env in the TZM-bl assay. Each graph shows neutralization by serum , purified IgG, and the flow through fraction at a 1:40 dilution (FT). RPz16, an RM from the same immunization group that lacked serum nAb activity, was also included in the analysis, as well as normal RM serum (NMS).(TIF)Click here for additional data file.S7 FigA vertical point plot depicts neutralization of four previously characterized HIV-1/SHIV Env PV: a clade C tier 1 HIV-1 Env (93MW965.26); two tier 2 HIV-1 Envs from a global panel (clade A 398F1 and CRF01_AE CNE55); and a SHIV Env (1157 PV) cloned from clade C SHIV1157pd3N4 challenge stock with a tier 2 phenotype. The clade C tier 2 SHIV1157pd3N4 challenge stock grown in RM PBMC (SHIV.1157) was also included. Z1800M and R66M T/F Env PV were included as the unknowns with respect to neutralization tier. Each Env PV/virus was tested for susceptibility to neutralization by CD4-induced epitope mAb 17b, HIVIG, V3 bnAb 447-52D, individual plasma samples from five Rwandan (R) and five Zambian (Z) HIV+ individuals, and pools of plasma from a group of Rwandan and Zambian HIV+ individuals. The graph depicts residual infectivity when each mAb was tested at 5 μg/ml and each plasma/serum sample diluted to 1:100. Lower values indicate greater susceptibility to neutralization. A two-way ANOVA with Dunnett’s correction for multiple comparisons was performed and revealed that all Envs, including the Z1800M and R66M T/F Envs, were statistically different from the tier 1 Env 93MW965.26 .(TIF)Click here for additional data file.S8 Fig(A) as well as positive control bnAb VRC01 (B) were tested in the TZM-bl assay against the Z1800M T/F Env, 5-month Envs D10 and D11, and Env chimeras in which the T/F Env contained fragments from D10 and D11 or mutants in which changes in V5 from D10 and D11 were introduced into the T/F Env.Negative controls including non-neutralizing serum from Z1800M T/F gp120 immunized animal RPz16 (wk 55 and wk63) and normal (naïve) RM serum (TIF)Click here for additional data file.S9 Fig(A) 64 mAbs were isolated from Z1800M at 7 months post-infection by sorting B cells with the autologous T/F Env gp120. The mAbs were tested for neutralization of the autologous T/F Env PV at a concentration of 10 μg/ml. Percent viral infectivity is shown on the y-axis relative to no test mAb. Three neutralizing mAbs from Z1800M are indicated by color. VRC01 and EM4C04 were used as positive and negative controls, respectively. (B) Genetic characteristics of the 3 Z1800M neutralizing mAbs are shown, using IMGT VQuest. mAbs 1A8 and 2H10 are considered clonally related (shaded gray). (C) A binning competition assay was conducted on the Octet Red96 between each of the Z1800M neutralizing mAbs and reference bnAbs VRC01, 3074, and PGT121, as well as the autologous 1A8. The percent residual binding of the reference mAb in the presence of the competitor is shown, with red indicating strong competition and blue representing weak or no competition.(TIF)Click here for additional data file.S10 Fig(A) The gating strategy used to isolate single B cells from cryopreserved week 63 PBMC that bound to Z1800M T/F gp120 but not the same protein when V5 was mutated is shown. (B) Live CD20+ IgG+ B cells that were positive for Z1800M T/F gp120-AF647 and negative for D11 mutated V5 gp120-PE were selected (upper left). (C) Germlines from all VH sequences isolated from single B cells selected for binding to Z1800M T/F gp120 (TF) or differential binding to the T/F but not the D11 V5 mutated protein (V5) are shown in donut plots. The total number of VH sequences is indicated. Enrichment of the neutralizing clonotype derived from IGHV4-79*02 is shown by the arrows.(TIF)Click here for additional data file.S11 FigComparison between BG505 and Z1800M T/F Env SOSIP models. Amino acid residues based on HXB2 are shown.(TIF)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


This variant was mainly prevalent among MSM, and was overrepresented in transmission clusters, suggesting that it could have driven the rapid expansion of CRF07_BC in MSM, though founder effects cannot be ruled out. It was descended from an evolutionary intermediate accumulating four specific mutations and formed an independent phylogenetic node with an estimated origin time in 2003. The K28E32 variant was demonstrated to have significantly higher in vitro HIV-1 replication ability than the wild type. Mutations E28K and K32E play a critical role in the improvement of in vitro HIV-1 replication ability, reflected by improved reverse transcription activity. The results could allow public health officials to use this marker  coding region) to target prevention measures prioritizing MSM population and persons infected with this variant for test and treat initiatives.HIV-1 CRF07_BC originated among injection drug users (IDUs) in China. After diffusing into men who have sex with men (MSM), CRF07_BC has shown a rapid expansion in this group; however, the mechanism remains unclear. Here, we identified a new KIMPORTANCE HIV-1 has very high mutation rate that is correlated with the survival and adaption of the virus. The variants with higher transmissibility may be more selective advantage than the strains with higher virulence. Several HIV-1 variants were previously demonstrated to be correlated with higher viral load and lower CD4 T cell count. Here, we first identified a new variant (the K28E32 variant) of HIV-1 CRF07_BC, described its origin and evolutionary dynamics, and demonstrated its higher in vitro HIV-1 replication ability than the wild type. We demonstrated that five RT mutations  significantly improve in vitro HIV-1 replication ability. The appearance of the new K28E32 variant was associated with the rapidly increasing prevalence of CRF07_BC among MSM. Since the first case was reported in 1985, HIV/AIDS has been a national problem in China, with 1,045,000 people living with HIV/AIDS by the end of 2020 . Second,5–CRF07_BC originated in early 1990s, and mainly circulated among injection drug users (IDUs) , 9. Afte28E32 variant was characterized by 5 specific amino patterns  at sites 28, 32, 248, 249, and 338 of reverse transcriptase (RT) coding region and was demonstrated to have higher in vitro HIV-1 replication activity than the wild type. Very high prevalence of the K28E32 variant among MSM and its overrepresentation in transmission clusters suggest that its appearance was associated with the rapid expansion of CRF07_BC among MSM.HIV-1 is mostly spread along contact networks with sexual or blood exposure risks , 18. NetRT coding region sequences (2289-3187nt in HXB2) from 1997–2013, eight large evolutionary (or transmission) clusters (ECs or TCs) were identified, consisting of 510 sequences (42.7%) . There were 350 (29.3%) non-cluster sequences, and the remaining 335 sequences formed small transmission clusters with <20 sequences. Of the 8 large ECs, 5 contained the sequences (n = 383) obtained from 2007 to 2013, and were named post-2007 clusters. The other 3 ECs included 127 sequences obtained during 1997 to 2012, and were named pre-2007 clusters. We then investigated whether there was a difference in signature residues between cluster and non-cluster sequences. We found 2 distinct amino acid sequence features that separated cluster and non-cluster sequences. The vast majority of cluster sequences had Lys (K) and Glu (E) residues at sites 28 and 32 of RT coding region (376/510: 73.7%) (https://www.hiv.lanl.gov/content/sequence/LOCATE/locate.html), respectively, and were defined as the K28E32 variant, while the vast majority of non-cluster sequences (269/350: 76.9%) had E and K residues at 28 and 32 sites, respectively, and were defined as the wild-type (WT) or the E28K32 strain   . Interes strains .28E32 variant carried other specific amino acids in RT using all available sequences of the entire CRF07_BC RT coding region. We found that the vast majority (92.4%) of the K28E32 variants carried 3 additional specific amino acids mutations E248V, K249Q, and T338S in the RT coding region  that carry 1 to 5 of the specific mutations and might originate via second-generation recombination between the CRF07_BC K28E32 variant and CRF01_AE (28E32 variant (Table S1).To test whether the 5 mutations are specific for CRF07_BC, we analyzed the amino acid characteristics at the 5 sites of the RT coding region of other HIV-1 subtypes and CRFs. The results showed that the representative strains of most analyzed subtypes and CRFs do not carry any one of the 5 specific mutations, except the KCRF01_AE . The preCRF01_AE . We furt28E32 variant, Bayesian phylogenetic analysis was performed. The origin time of CRF07_BC was estimated to be 1993.6 , very close to the earlier estimates (28E32 variants form a large independent clade that is located at the tip of the tree. The time to the most recent common ancestor (tMRCA) of the K28E32 variants was estimated to be 2003.0 (95% CI: 2001.2–2004.4) , indicating that the variant was formed since 2003. The earliest circulating K28E32 variant was detected in 2006, about 3 years later since its origination.To trace the origin and evolutionary history of the Kstimates , all the28E32 variant clade with the wild-type strains, suggesting that it was an evolutionary intermediate from the WT strain to the K28E32 variant. The intermediate was isolated from a man who had sex with men in 2010 and featured by K-E-VQ-T at 28, 32, 248/249, and 338 sites of the RT coding region, respectively (28E32 variants was estimated to be 2000.8 (95% CI: 1998.3-2002.9), and the divergence time of the intermediate from the WT strains was estimated to be 1998.3 (95% CI: 1996.2-2000.2) . These suggest that the origin of the K28E32 variant experienced at least 2 evolutionary steps and in the evolutionary events, 4 mutations E28K, K32E, E248V, and K249Q were first fixed during 1998 to 2000, and then T338S was fixed during 2000 to 2003.One sequence (green branch in the MCC tree) carrying mutations E28K, K32E, E248V and K249Q was identified to link the Kectively . The tMA28E32 variant and the wild-type, there are several variants carried 1 to 4 mutations at the 5 specific sites. Three variants carrying any 1 or 2 of mutations E28K and K32E were found in the clade of the wild-type strains , and 2 variants carrying one other mutation (V248A or Q249H) were found  of analyzed ML trees . When thin clade . These relection , indicat28E32 variant of CRF07_BC using Bayesian skyline plot analysis. The wild-type strain experienced a continuous expansion since its origin in early 1990s, and peaked in about 2005, 2 years after the generation of the K28E32 variant  of CRF07_BC among different high-risk groups. We found that the K28E32 variant mainly appeared among MSM (73.2%), whereas the wild-type strain was mainly prevalent among IDUs (84.8%) (28E32 variant was significantly higher among MSM (87.4%) than IDUs (3.7%) and the heterosexuals (23.8%) (P < 0.0001 for both). Among the heterosexuals, the K28E32 variant and wild-type strain accounted for 23.8%, and 61.9%, respectively  . It is nectively . These r28E32 variant and the wild-type strain among IDUs, MSM, and the heterosexuals during the past decades using all available sequences  before 2007 to 70.4% in 2019–2020 among MSM .The crystal structure shows the RT enzyme of HIV-1, like a human right hand, contains 4 subdomains: fingers (1–85 and 118–155), palm (86–117 and 156–236), thumb (positions 237–318), and connection (319–426) . Mutatio28E32 variant on HIV-1 replication, we constructed the infectious clones of the K28E32 variant (NL4-3_07RT-K28E32) and the wild-type strain (NL4-3_07RT-WT) by incorporating their RT coding fragments into the full-length HIV-1 NL4-3 molecular clone. Infectious virions were generated in HEK293T cells by transfection. Normalized amounts (10 ng of p24) of the K28E32 variant and the wild-type virions were used to infect MT-2 cells. Viral replication was monitored over a period of 12 days by quantifying p24 and viral RNA copies in the culture supernatant. Both NL4-3_07RT-K28E32 and NL4-3_07RT-WT showed consistent replication dynamics. HIV-1 RNA and p24 levels continuously increased, especially during 6–10 days after infection (28E32 generated significantly higher HIV-1 RNA and p24 levels than NL4-3_07RT-WT (P < 0.01), suggesting that the K28E32 variant has greater in vitro replication capacity than the wild-type strain.To determine the influence of the Knfection . HIV-1 R28E32 variant has 5 specific mutations. To investigate the crucial mutations influencing HIV-1 replication, we further constructed 6 additional mutants  and second-strand transfer (late RT). The early and late RT products of the wild-type strain peaked at 2 and 4 h postinfection, respectively, and then slowly decreased  to d. Coectively  to d. Co28E32 variant, MUT-4 and MUT-1 had significantly higher 2LTR products than the wild-type strain; while in contrast, MUT-3 exhibited substantially lower amount of 2LTR products. Higher level of 2LTR products by the K28E32 variant may be simply attributed to higher accumulation of late RT products that enhances the pre-viral DNA nuclear translocation.We also examined the ability of various RT mutants for nuclear localization by quantifying 2LTR circle formation. 2LTR products continuously increase up to 48 h postinfection for WT and all mutants . Analysiin vitro replication ability and is mainly circulating among MSM.HIV-1 is one of the most variable RNA viruses with high mutation rate and recombination potential caused by the error-prone nature and the template-jump mechanism of RT enzyme in HIV-1 replication, respectively –25. High–26–28E32 variant of HIV-1 CRF07_BC that has higher in vitro replication ability than the wild type. The finding and identification of the K28E32 variant suggest that transmission network analysis can also be used as a robust tool to find and identify newly emerging highly adapted variants. Given substantial effects in reducing HIV-1 transmission among high-risk groups such as MSM, transmission network analysis has been incorporated into the national guidelines for the routine monitoring and intervention of HIV-1 transmission by China CDC since 2021. National implementation of transmission network monitoring will benefit the finding of newly emerging HIV-1 variants overrepresented in transmission clusters in the future.HIV-1 exists in quasispecies with one or more mutations in host, and only one or few HIV-1 founder (or fitness) variants can be effectively transmitted from one host to another under strong transmission bottleneck , 34. In 38–https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/crfs.comp)  , 6. CRF0fs.comp) , 40. CRFfs.comp) , 44, 45.fs.comp) , 46, 47.fs.comp) , 13. Acc28E32 variant of CRF07_BC accounted for a significantly higher proportion among MSM than IDUs (P < 0.01), and may be responsible for the rapid expansion of CRF07_BC among MSM , and from the intermediate to the K28E32 variant. Of 5 specific mutations in the K28E32 variant, mutations E28K and K32E play a crucial role in enhancing in vitro replication capacity of the RT enzyme. The intermediate (MUT-4) had accumulated 4 of the 5 specific mutations, except T338S, and exhibited slightly lower level of in vitro HIV-1 replication than the K28E32 variant, but significantly higher level than the wild type. Because the full-length genomic sequence of the intermediate is not available, any differences in other genes between the intermediate and the K28E32 variant remains unclear. The appearance of the K28E32 variant to replace the evolutionary intermediate and the wild-type among MSM might be simply attributed to its stronger replication ability, a founder effect and/or the accumulation of additional adaptive mutations in other genes  than the wild-type (0.033) . The evolutionary rate of the K28E32 variant was estimated to be 1.781 × 10−3, also substantially lower than that (3.945 × 10−3) of the wild type. The 5 mutations in the RT coding region are specific features to define the K28E32 variant, and are involved in its origin; however, they were not subject to significantly positive selection. In view of the in vitro replication advantage conferred by the 5 specific mutations, selective sweep might contribute to the stability of the 5 specific residues and the lower genetic diversity of the K28E32 variant  sequences from the HIV database. There were 8 K28E32 variants and 36 wild-type strains. All these sequences, regardless of the K28E32 variant and the wild-type, were predicted to have CCR5 tropism using geno2pheno and the R5-X4 pred tool  of the K28E32 variant also carried specific mutations (data not shown). HIV-1 accessory proteins not only play crucial roles in HIV-1 replication, assembly, and survival, but also counteract host restriction factors   , 59. Whe28E32 variant that carries 5 specific mutations in the RT coding region, and exhibits higher in vitro HIV-1 replication ability than the wild type. Extremely high prevalence of the K28E32 variant among MSM and its overrepresentation in large transmission clusters suggest its association with the rapid expansion of CRF07_BC among MSM in recent years. The emergence and subsequent predominance of the K28E32 variant among MSM could be ascribed to its higher in vitro replication ability and/or simply a founder effect of this variant being propagated among groups that are currently being infected in China  on December, 2015. After removing those without geographic origin and sampling year, 1195 pol sequences (899 nt with a location of 2289–3187 nt in HXB2) were used for transmission cluster and evolutionary analyses. A ML tree was constructed using FastTree version 2.1 (http://meta.microbesonline.org/fasttree/), and HIV-1 evolutionary (or transmission) clusters were identified using ClusterPicker 1.2.1 with parameters of initial threshold: 0.9, main support threshold: 0.9, genetic distance threshold: 4.5 (http://weblogo.berkeley.edu/logo.cgi). Significance was evaluated using Viral Epidemiology Signature Pattern Analysis (VESPA: https://www.hiv.lanl.gov/content/sequence/VESPA/vespa.html). Viral tropism was determined using geno2pheno and the R5-X4 pred tool  in September, 2021.A total of 570 p51 (RT coding) sequences of CRF07_BC strains with known demographic information  were subjected to phylogenetic reconstruction using approximate maximum likelihood with PhyML 3.0 program. Among them, 207 sequences were from the newly diagnosed HIV-1-positive patients in Yunnan province and Shenzhen city from the year 2010 to 2016 in this study, who were participants in the National Key S&T Special Projects on Major Infectious Diseases. All participants signed written informed consents prior to sample collection, and completed standardized questionnaires that included demographic data. This study was reviewed and approved by the ethics committees of the Beijing Institute of Microbiology and Epidemiology. The other 363 sequences, which were sampled in China from 1997 to 2018, were downloaded from HIV database (https://itol.embl.de/).The GTR + G+I nucleotide substitution model was selected by using Smart Model Selection (SMS) . The heu8 length of chain sampling frequency of 1000  . After r of 1000 . All phyhttp://datamonkey.org), including single-likelihood ancestor counting (SLAC), mixed effects model of evolution (MEME), fixed effects likelihood (FEL), and fast, unconstrained Bayesian approximation (FUBAR), were used to identify positively selected sites in the RT coding region of CRF07_BC  containing 10% fetal bovine serum (FBS) (Gibco) and 100 units/mL penicillin and 100 μg/mL streptomycin. MT-2 cells were cultured in RPMI 1640 medium (Gibco) containing 10% FBS and 100 units/mL penicillin and 100 μg/mL streptomycin.HQ215552) . The infectious clone plasmid was linearized by restriction endonuclease digestion and purified by ApaI/EcoRI extraction. To generate the infectious clones of various CRF07_BC RT variants (mutants), the Q5 site-directed mutagenesis kit (NEB) was used to introduce corresponding substitutions into the recombinant CRF07_BC infectious clone. The substitution sites were confirmed by PCR and Sanger sequencing. A total of 7 CRF07_BC RT variants were constructed, including the K28E32 variant (MUT), and 6 related mutants (MUT-1-MUT-6). The characteristic amino acid sites of the CRF07_BC wild-type (WT) strain, K28E32 variant (MUT), and 6 related mutants (MUT-1-MUT-6) are listed in In order to obtain a recombinant CRF07_BC infectious clone, the RT coding region (HXB2:2550-3870) of HIV-1 subtype B infectious clone pNL4-3 was replaced by a RT coding fragment from a CRF07_BC strain (Accession Number: The recombinant CRF07_BC plasmids were transfected into HEK293T cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific) to generate virus stock. Culture supernatants were collected at 48 to 72h posttransfection. Infectious virions were detected by tissue culture infectious dose 50 (TCID50). HIV-1 p24 antigen expression was detected by enzyme-linked immune sorbent assay (ELISA). The correction of the mutations in generated mutant virions was further confirmed by RT-PCR and Sanger DNA sequencing using the RNA from the supernatant.5 MT-2 cells were infected with the viral supernatants containing 10 ng p24 antigen. After 6 h of incubation, the cells were washed twice with PBS, and fresh medium (RPMI 1640 containing 10% FBS) was added to each well. Infected cells were maintained at 37°C with 5% CO2 and the supernatants were collected at the indicated time points of 0, 2, 4, 6, 8, 10, and 12 days after infection. The p24 antigen content in the supernatant was detected by ELISA. Viral RNA was extracted from the supernatant using Viral RNA minikit (QIAamp) and a previously established RT-qPCR assay was performed to determine the mRNA copies in the viral supernatants . HIV-1 replication intermediates  were measured by qPCR assays as previously described . The priThe qPCR assays were performed by using the GoldStar Probe Mixture (CoWin Biosciences). A 15 μL qPCR system was set up, containing 1 × gold star TaqMan mixture, 0.2 μM (each) forward and reverse primers, 0.2 μM probe, and 500 ng template DNA or non-template control (NTC). The reactions were performed using LightCycle 480 (Roche), and the reaction condition was pre-denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15s, and annealing and extension at 60°C for 1 min.unpaired t test or One-Way ANOVA with Tukey’s multiple-comparison test. Data are presented as means ± SD or as described in the corresponding legends.All data were analyzed using the GraphPad Prism software. Statistical evaluation was performed by Student’s pol sequence alignments were available at https://github.com/mayingying1997/CRF_07BC-sequence.git. The sequences obtained in this study were submitted to GenBank and the accession numbers are ON241448-ON241654. Other sequences used in this study were downloaded from GenBank. All the software used in this study are available from open source.The