A greater proportion of pregnant women with COVID-19 have mild disease compared with their non-pregnant counterparts. Paradoxically, however, they are at higher risk of developing severe disease, requiring respiratory support and admission to intensive care. The delta SARS-Cov-2 variant is associated with increased risk of hospitalization and morbidity in unvaccinated pregnant populations. However, it is not known whether the worse pregnancy outcomes associated with the delta variant are due to a direct effect of the virus on the pregnancy, or whether this effect is mediated through more severe maternal infection. Here, we synthesize studies of COVID-19 pregnancies, focusing on the different routes of SARS-CoV-2 infection of lung and placenta, and the mechanisms of syncytial formation for each SARS-CoV-2 variant. To delineate COVID-19 complications in pregnant women, future studies should explore whether the delta variant causes greater placental infection compared to other variants and contributes to increased syncytial formation. Virology; Pregnancy Although pregnant women with COVID-19 usually display mild to moderate symptoms when compared to non-pregnant women with the same risk profile, they are at higher risk of developing severe disease, requiring respiratory support and admission to intensive care (ICU) . The USAWhat is not known, however, is whether the worse pregnancy outcomes associated with the delta variant are due to a direct effect of the virus on the pregnancy, or whether this effect is mediated through more severe maternal infection. The delta variant has been associated with greater viral loads than the alpha variant , but theA national UK prospective cohort study showed that, after adjusting for pre-existing medical conditions and sociodemographic variables, the proportion of symptomatic pregnant women hospitalized with moderate to severe COVID-19 increased significantly from the wild type (24%) to alpha (36%) to the delta period (45%) . FurtherA meta-analysis evaluated the severity of disease caused by the SARS-COV-2 variants of concern (VOCs) in the general population from June 1, 2020 to October 15, 2021. This showed that the beta and delta variants pose a greater risk of hospitalization, ICU admission, and mortality compared to the alpha, gamma, and wild-type variants . FindingFull vaccination during pregnancy is essential but data are limited regarding transplacental antibody transfer after vaccination with the currently available vaccines. A small study (n = 29) evaluating maternal and umbilical cord blood on the day of birth assessed neutralizing antibody levels for both the wild type and delta variant and showed a pronounced reduction for the delta variant .Although vertical transmission of SARS-CoV-2 is rare, has been observed  and placPregnant women with gestational diabetes mellitus are nine times as likely as those without diabetes to be infected with the delta variant and are three times more susceptible than those with cardiovascular disease or hypertension . Women wIn a review, Rangchaikul and Venketaraman suggested some parameters that may predispose the pregnant patient to greater susceptibility to and severity of SARS-CoV-2. These include, among others, hampering of cell-mediated immune clearance of SARS-CoV-2, altered immunomodulation by progesterone, coagulation, and complement-associated hyperinflammation .A recent report described two cases of intra-uterine fetal demise and a case of severe fetal distress after infection of unvaccinated mothers with the delta variant and mild COVID-19 disease severity . All motCell-to-cell fusion that leads to the formation of a syncytium is a physiological mechanism that occurs in various cell types, such as myocytes. The same mechanism allows viruses to infect neighboring cells without the exocytosis of free virus, and virus-infected syncytia increase tissue damage. Infectious syncytia are formed by the attachment of virions to cells or cell-to-cell fusion and are typical of coronaviruses . FormatiThe rate of formation of syncytia by SARS-CoV-2 has been shown to be much faster than by SARS-CoV-1 . Of notein vitro. Of the cells positive for any variant, at least 43% were distributed as clusters of 2 or more cells, and the delta variant proved to have the highest percentage (>78%) of infected cells organized in clusters, relative to alpha (ca. 59%) or beta (ca. 69%) variants  . Anothervariants .There is evidence, therefore, that the delta variant spike protein may increase cell-to-cell fusion  when comPlacentation is a complex multi-step process that enables placental perfusion, during which trophoblast cells adhere, invade, and remodel spiral arteries. Using a process called syncytialization, the cytotrophoblasts fuse with the overlying giant multinucleated syncytiotrophoblasts and form the outer layer of the placental microvilli  renderinThere may be small amounts of fibrin deposition in normal placentas, which is increased in HDP . MassiveFor a cell to be permissive to SARS-CoV-2, it must express the angiotensin-converting enzyme 2 (ACE2) receptor and have protease activity—transmembrane serine protease 2 (TMPRSS2) . After tACE2 and TMPRSS2  co-expressed ACE2/TMPRSS2 (Other studies have assessed the expression of ocytosis . ACE2 anocytosis . Another/TMPRSS2 .ACE2 (but not TMPRSS2 or Furin) and the protein levels of IFITM1 and IFITM3 were higher in women with severe COVID-19  and CTSL, which, according to the authors  syncytial aggregates in the maternal circulation. Syncytiotrophoblast-derived syncytial knots, which become multinucleate syncytial aggregates that express the antiangiogenic protein sFlt-1, have previously been detected in the lungs of pregnant women, and the number of syncytial aggregates in the maternal lungs was higher in women with preeclampsia . This prInfection by the SARS-CoV-2 delta variant in pregnant women might induce increased pathogenicity via epigenomic regulation or other non-canonical entry routes. Future research should focus on comparison of the alveolar and placental barriers, cell-to-cell communication, and the cellular and molecular partners involved.Key to syncytialization is the hypomethylation  and exprUnlike the alveolo-capillary barrier in the lungs, the syncytiotrophoblast expresses little or no caveolin-1  or caveoIntercellular communication via gap junctions is also necessary for trophoblastic cell fusion and syncytiotrophoblast formation. The involvement of the gap junction protein connexin-43 was immunodetected at the intercellular boundaries between aggregated cells; the expression disappeared after cellular fusion . There iFinally, the extensive syncytial formation due to SARS-CoV-2 infection might contribute to increased viral dissemination and cause greater and more extensive placental tissue damage . MoreoveThe definitions of the severity of COVID-19 may vary among investigators. The data presented in this review are limited by the increasing rate of vaccination among pregnant women, the small number of published studies and preprints, and in some cases, the small sample size analyzed and the limited sequencing results in the studies of the pregnant patients.During the delta variant wave of the COVID-19 pandemic, the rate of admission of pregnant women to hospital and to ICU was increased compared to the first wave and the alpha wave of the pandemic. This could be due to various variant-specific factors, such as the increased delta variant transmissibility compared to the previous variants. Moreover, the significantly greater respiratory viral load of delta and its ability to form syncytia in the lungs could cause more severe disease and result in hypoxia. In addition, other patient-specific factors could be the lower vaccination rates in pregnant women compared to the general population, and pregnancy itself conferring greater susceptibility. Studies of changes in the epigenomic landscape and transcriptome, together with studies of placental histology, should address whether the delta variant causes greater placental infection and contributes to increased syncytia formation. This type of analysis would delineate whether the increased incidence of adverse pregnancy outcomes seen with the delta variant is due to more severe systemic maternal infection or due to a direct effect of this variant on the fetus or placenta.Data for this Review were identified by searches of Pubmed/Medline, Current Contents, BiorXiv, and references from relevant articles and preprints, using the search terms “pregnancy”, “COVID-19”, “delta variant”, “omicron”, and “syncytia”.


In the clinical fitting of cochlear implants (CIs), the lowest input acoustic frequency is typically much lower than the characteristic frequency associated with the most apical electrode position, due to the limited electrode insertion depth. For bilateral CI users, electrode positions may differ across ears. However, the same acoustic-to-electrode frequency allocation table (FAT) is typically assigned to both ears. As such, bilateral CI users may experience both intra-aural frequency mismatch within each ear and inter-aural mismatch across ears. This inter-aural mismatch may limit the ability of bilateral CI users to take advantage of spatial cues when attempting to segregate competing speech. Adjusting the FAT to tonotopically match the electrode position in each ear  is theorized to reduce this inter-aural mismatch. Unfortunately, this approach may also introduce the loss of acoustic information below the modified input acoustic frequency. The present study explored the trade-off between reduced inter-aural frequency mismatch and low-frequency information loss for segregation of competing speech. Normal-hearing participants were tested while listening to acoustic simulations of bilateral CIs. Speech reception thresholds (SRTs) were measured for target sentences produced by a male talker in the presence of two different male talkers. Masker speech was either co-located with or spatially separated from the target speech. The bilateral CI simulations were produced by 16-channel sinewave vocoders; the simulated insertion depth was fixed in one ear and varied in the other ear, resulting in an inter-aural mismatch of 0, 2, or 6 mm in terms of cochlear place. Two FAT conditions were compared: 1) clinical (200–8000 Hz in both ears), or 2) matched to the simulated insertion depth in each ear. Results showed that SRTs were significantly lower with the matched than with the clinical FAT, regardless of the insertion depth or spatial configuration of the masker speech. The largest improvement in SRTs with the matched FAT was observed when the inter-aural mismatch was largest (6 mm). These results suggest that minimizing inter-aural mismatch with tonotopically matched FATs may benefit bilateral CI users’ ability to segregate competing speech despite substantial low-frequency information loss in ears with shallow insertion depths. In the clinical fitting of cochlear implants (CIs), the lowest acoustic input frequency is typically much lower than the characteristic frequency associated with the most apical electrode position , resultiintra-aural tonotopic mismatch in each ear, bilateral CI users may also experience inter-aural mismatch, where the same input frequency stimulates a different cochlear place across ears. Inter-aural mismatch has been shown to limit binaural performance and benefits in bilateral CI users [For bilateral CI users, the electrode position may differ across ears. However, during the clinical fitting process, the same FAT is typically assigned to these electrodes in each ear. As such, in addition to the CI users , 8 and iCI users , 10. FurCI users , 11, whiCI users  and speeCI users , 10.Symmetrically placed speech maskers have been used to estimate spatial masking release, relative to co-located target and masker speech. These spatial configurations may be implemented in sound fields via loudspeaker presentations or in virtual space via a modified headphone presentation. In the case of a headphone presentation, the difference between diotic and dichotic masker presentation have been used to estimate spatial masking release . HoweverSince it is difficult to estimate the functional spectral resolution and degree of intra- and inter-aural mismatch in bilateral CI users, acoustic simulations of bilateral CIs in NH listeners have been used to parametrically control the spectral resolution and the electrode position in each ear. Such bilateral CI simulations necessitate the headphone presentation style to deliver the signal processing independently to each ear. In a previous study, Xu et al.  used dioIn Xu et al. , the degAs noted above, adjusting the FAT to match the characteristic frequency associated with the electrode position may reduce intra- and inter-aural mismatch, but also introduces low-frequency information loss in one or both ears. The amount of low-frequency information loss largely depends on the insertion depth, with shallower insertion depths resulting in larger low-frequency information loss. For unilateral CI users with a shallow insertion depth, adjusting the FAT may not be an ideal approach, as the deficits associated with low-frequency information loss tend to overshadow the benefits of reducing the intra-aural mismatch. However, for bilateral CI users with different insertion depths across ears, the greater low-frequency information loss in the CI ear with the shallower insertion may be less detrimental to bilateral speech understanding, as there will be less information loss in the ear with the deeper insertion. The tradeoff between tonotopic matching and information loss has not been widely studied in bilateral CI users or in NH participants listening to acoustic simulations of bilateral CIs. Moreover, it is unclear how tonotopic matching may help bilateral CI users take advantage of spatial masking cues. While bilateral CI users receive some of the benefits that come with binaural hearing, they often have difficulty with binaural fusion, i.e., fusing sounds from the two ears into a single percept . ReducinThe goal of the present study was to evaluate the effects of tonotopic matching on the segregation of competing speech, where target and masker speech was either co-located or spatially separated. NH listeners were tested while listening to acoustic simulations of bilateral CIs via headphones. Unlike the diotic-dichotic masker presentation in Xu et al. , HRTFs wThis study was approved by the Institutional Review Board of the University of California, Los Angeles (UCLA IRB#19–000722) and this research was conducted in accordance with the principles of the Declaration of Helsinki and its later amendments. Prior to participation, written informed consent was obtained from all participants, in accordance with a protocol approved by the Institutional Review Board at the University of California, Los Angeles. All participants were given the opportunity to ask questions pertaining to the safety and modality by which the study would be conducted.Twenty NH adults  participated in the study. All participants had pure tone thresholds < 25 dB hearing loss (HL) at all audiometric frequencies between 250 and 8000 Hz in both ears. The participants were a random cohort with no known affiliations between any subjects and all participants were compensated upon completion.Speech reception thresholds (SRTs), defined as the target-masker ratio (TMR) that produces 50% correct recognition of keywords in competing speech, were measured using a closed-set, matrix-styled test paradigm . Target All target and masker stimuli were generated in real-time and delivered to circumaural headphones (Sennheiser HDA 200) via an audio interface (Edirol UA-25EX) connected to a mixer (Mackie 402). Non-individualized HRTFs were also used to create a virtual auditory space for headphone presentation of the stimuli . The tarIn each test trial, the target and masker sentences were first generated according to the specified TMR. The TMR was calculated according to long-term root-mean-squared (RMS) power between the target sentence and masker sentences. The target and masker sentences were first processed by the HRTF and then mixed independently into the left and right channel. The mixed target and masker sentences in each channel were then processed by a 16-channel sine-wave vocoder . First, John”) and then click on one of the 10 response choices for each of the Number and Color categories; no other selections could be made from the remaining categories, which were greyed out. The target level was fixed at 65 dBA. The TMR was adjusted by varying the masker level from trial to trial according to the correctness of the responses for the target Number and Color keywords; the initial step size was 4 dB for the first two reversals in TMR, and the final step size was 2 dB. The SRT was calculated by averaging the last six reversals in TMR. The 12 listening conditions  were randomized within a test block and 2–3 blocks were tested for each participant; SRTs for each condition were averaged across blocks. Participants were given no practice or previews prior to the testing sessions, and no feedback was provided during testing. All testing was completed in a single session with short breaks between test blocks.Testing was performed in a sound-attenuating booth. SRTs were measured using an adaptive procedure (1-up/1-down) that produced a 50% correct sentence recognition. The procedure was similar to a coordinate response matrix (CRM) test , 24. ParSRT data were analyzed using a linear mixed model (LMM), with FAT, spatial configuration type, and inter-aural mismatch, as fixed effects and participant as a random effect. The improvement in SRT data were also analyzed using a LMM, with benefit condition and inter-aural mismatch as fixed effects, and participant as a random effect. For the LMMs, significance was established for p < 0.05. Bonferroni correction was applied to post-hoc pairwise comparisons. LMMs were performed using SPSS . All figures were generated using Sigmaplot software (Version 14).The present bilateral CI simulation data show that binaural SRTs worsened with increasing inter-aural mismatch for both co-located and spatially separated target and maskers , Panel AWith 0-mm inter-aural mismatch, the mean SRT for the co-located maskers was 9.8 dB, similar to Xu et al. . Mean SRWith the spatially separated maskers, SRTs worsened by 3.2 dB with 2-mm inter-aural mismatch, much less than the drop of 7.5 dB for the dichotic masker presentation in Xu et al. . This isFor the clinical FAT, the intra-aural tonotopic mismatch increased as the insertion depth was reduced in the right ear. As shown in For the co-located maskers, mean SRTs with the matched FAT were 7.3, 8.1, and 8.7 dB TMR for the 0-mm, 2-mm, and 6-mm inter-aural mismatch conditions, respectively, much lower than SRTs with the clinical FAT. Tonotopic matching within each ear improved mean SRTs by 2.6, 3.5, and 5.0 dB as the inter-aural mismatch was increased from 0 to 2 to 6 mm, respectively. These results suggest that, when tonotopically matched within each ear, spectral cues from the ear with the shallower insertion depth might not interfere with the recognition of target speech in the ear with the deeper insertion, despite the greater information loss.With the spatially separated maskers, mean SRTs with the matched FAT were 5.4, 7.1, and 7.6 dB TMR for the 0-mm, 2-mm, and 6-mm inter-aural mismatch conditions, respectively. As with the co-located maskers, SRTs with spatially separated maskers were much lower with the matched FAT than with the clinical FAT. Tonotopic matching within each ear improved mean SRTs by 2.6, 4.2, and 4.4 dB as the inter-aural mismatch was increased from 0 to 2 to 6 mm, respectively. There was no significant difference in the benefit of tonotopic matching within each ear when maskers were co-located or spatially separated.While the benefit of spatial cues was similar with the clinical and matched FATs, the benefit of tonotopic matching was much larger than the benefit of spatial cues top of . This liThe bilateral CI simulation data collected in this study are consistent with Svirsky et al. , who shoThere are some limitations and caveats to the present study that should be considered. First, the NH participants presumably had uniform nerve survival within the carrier frequency ranges tested. This is not likely to be the case in real bilateral CI users; even when there is no difference in electrode position across ears, there may be uneven nerve survival patterns within each ear, resulting in inter-aural mismatch in terms of the electrode-neural interface. Second, performance was acutely measured in the present NH listeners with no training, feedback, or practicing experience with the various CI simulations. In the case of real CI users, long-term experience may mitigate some of the effects of intra- and inter-aural frequency mismatch due to neuroplastic adaptation over time. However, it still may be advantageous to reduce inter-aural mismatch in bilateral CI users, especially during the initial usage period with the device . Thus, iThe present study explored the trade-off between reduced intra-aural frequency mismatch and the loss of low-frequency information for the segregation of competing speech in normal-hearing (NH) participants listening to acoustic simulations of bilateral CIs where differences in insertion depth were varied. Major findings include:The matched FAT provided better SRTs than the clinical FAT, regardless of the insertion depth or spatial configuration. This suggests a strong benefit for tonotopic matching within and across ears.Compared to the clinical FAT, the matched FAT provided the largest improvement in SRTs when there was a large difference in insertion depth (6 mm). Moreover, spatial masking release significantly depended on the difference in insertion depth across ears for the clinical FAT, but not for the matched FAT.Minimizing intra-and inter-aural mismatch by adjusting the FAT may benefit overall SRTs, especially when there is a large difference in insertion depth across ears. Minimizing intra-aural mismatch may also improve masking releases when there is a moderate difference in insertion depth across ears.These tonotopic matching benefits were observed despite substantial low-frequency information loss in the CI ear with a very shallow insertion depth. Estimates of electrode positions in each ear may guide the implementation of optimal FATs that can improve the ability of bilateral CI users to segregate competing speech with or without spatial cues.S1 Data(XLSX)Click here for additional data file.


The rise in human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) has prompted a quest for further understanding of the role of high-risk HPV in tumor initiation and progression. Patients with HPV-positive OPSCC (HPV+ OPSCC) have better prognoses than their HPV-negative counterparts; however, current therapeutic strategies for HPV+ OPSCC are overly aggressive and leave patients with life-long sequalae and poor quality of life. This highlights a need for customized treatment. Several clinical trials of treatment de-intensification to reduce acute and late toxicity without compromising efficacy have been conducted. This article reviews the differences and similarities in the pathogenesis and progression of HPV-related OPSCC compared to cervical cancer, with emphasis on the role of prophylactic and therapeutic vaccines as a potential de-intensification treatment strategy. Overall, the future development of novel and effective therapeutic agents for HPV-associated head and neck tumors promises to meet the challenges posed by this growing epidemic. Oropharyngeal squamous cell carcinoma (OPSCC) arises most commonly in the palatine tonsils or the base of the tongue. As with other squamous cell carcinomas f the head and neck, it is classically caused by exposure to extrinsic carcinogens such as smoking and alcohol . Since tThe discovery of the etiological role of HPV during OPSCC tumorigenesis is closely linked to research of cervical cancer of the uterus. The first reports pointing to the potential role of HPV in OPSCC were based on epidemiological studies demonstrating a correlation between the relative incidence of cancers of the cervix and cancers of the oropharynx in certain geographical areas ,11. MuchAlthough cervical cancer and OPSCC have HPV as a common etiology, their respective tumors exhibit distinct epidemiological and transmission patterns . First, It has been shown that high-risk HPV types are a major risk factor for cancer development. There are 15 high-risk HPV genotypes . Among these 15 high risk HPV subtypes, the HPV16 and HPV18 genotypes cause approximately 70% of cervical cancers. The other subtypes are known to be carcinogenic in a lower proportion. For OPSCC, the HPV16 is the main subtype associated with more than 90% of the non-smoking-related cases [HPV is transmitted sexually, and the spectrum of HPV-related cancers is associated—though not exclusively—with sexual practices, including vaginal and anal intercourse, hand-to-genital contact, oral sex, and/or the use of fomites  . The risIt has been demonstrated that, after inoculation of the virus, the HPV infection in the cervix resolves in more than 90% of cases after two years, while only 50% of women undergo seroconversion . For HPVFor cervical cancer, longitudinal studies have shown that cervical intraepithelial neoplasia (CIN) can occur after a few months . Upon inFor OPSCC, the steps from infection to invasive cancer are not well understood. Further, since it is thought that infection occurs in the crypts of the palatine and base-of-tongue tonsils, the observation of the steps of carcinogenesis are rendered arduous . As we wThere are over 150 HPV strains (of which 15 are high-risk). HPV is a double-strand DNA virus measuring about 8000 base pairs . Each geTo infect the host cells, the virus binds to the basement membrane, which in turn activates the L2 protein (a minor capsid protein) via furin-mediated cleavage . This exHPV DNA provides multiple open reading frames (ORF), designated as early (E) or late (L). E1 is a DNA replication enzyme essential for the amplification of the viral episome, while E2 is implicated in virus replication, transcription, and genome division during the viral life cycle. The structural proteins L1 and L2 are for the shell of the virus, as discussed above. The E3 ORF is lacking due to a sequencing error. The remaining E4 and E5 are involved in cell cycle entry, immune evasion and virus release . HPV hasThe HPV virus joins to the keratinocyte at the start of its journey and replicates in a cell that may die of natural causes; however, the virus does not induce necrosis per se, thus limiting inflammation and presentation of antigens to the immune system. It stays in the epithelium, thus avoiding a blood-borne or viremic phase . An addiAn HPV infection leading to cancer is the result of persistent infection and E6/E7 oncogene expression. This is the least common manifestation of HPV. Most infections are subclinical without any sequelae. Data indicate that viral DNA integration to the cell genome occurs frequently in cervical cancer, as episomal viral DNA can be successfully suppressed by IFNb-induced immune response . PremaliAs discussed above, E6 and E7 proteins are crucial for HPV-driven carcinogenesis. It is known that HPVs exert their oncogenic role after DNA integration, gene expression of E5, E6 and E7 loci and p53/pRb host protein suppression, leading to increased cell proliferation and contributing to carcinogenesis. The E6 protein is a 150-amino-acid zinc-finger-bearing polypeptide. These zinc fingers are responsible for the enzymatic activity of E6. The primary interaction seen with E6 is to bind an alpha helical acidic LXXLL peptide . ThroughAside from its main role in binding and degrading P53, a plethora of functions of the E6 proteins have been described . E6s froIn differentiated keratinocytes, cellular DNA replication is switched off, meaning that high-risk HPV types have to stimulate replication in a more unnatural state. For this purpose, the role of E7 seems crucial to modulate the induction of DNA synthesis . HPV E7 A further difference between high- and low-risk HPV types involves alternative splice variants. High-risk HPV transcription patterns involve a number of alternative splices which generate a complex pattern of mRNAs . AlthougInterestingly, it seems that regulation of alternative splice variant E6* is dependent upon extracellular stimuli. In particular, E6* splicing is dependent on the presence of EGF and is mediated by the MAP kinase MEK1/2 pathway. On the other hand, it has been reported that E6* is able to inhibit the degradation of p53 by E6. Moreover, alternative splicing of E6 facilitates access to the E7 start codon and its translation. Hence, as a further consequence of E6 exon exclusion, higher levels of E7 protein are observed . Taking Prophylactic vaccines are directed against the L1 surface protein of the different HPV serotypes and can prevent primary infection of the cervix and tonsils. For cervical cancer, they lower the re-infection/recurrence rate after treatment for LSIL-HSIL. In contrast, therapeutic vaccines are directed against E6 and/or E7 proteins, expressed by HPV-induced invasive cancer of the cervix and oropharynx.Experimental studies have shown that virus-like particles induce a strong response in both the adaptive and the innate immune systems, with robust generation of neutralizing antibodies ,45. The Today, several prophylactic HPV vaccines have been commercialized . They arVaccination before puberty is now recommended in most Western countries . It is eSeveral randomized controlled trials have been conducted in non-vaccinated women with cervical intraepithelial neoplasia (CIN), investigating the efficacy of vaccination in reducing the risk of recurrence/second primary tumor. In the SPERANZA project, women were randomly assigned to HPV vaccination after having being treated for CIN 2 with a loop electrosurgical excision procedure . The recSince HPV vaccines prevent virus inoculation by producing antibodies against L1 protein and given the fact that L1 is not expressed during the oncogenic process, it is thought that the reduction in recurrence risk is due either to prevention of new inoculation (possibly by another strain) or to auto-inoculation from episodic dormant viral activation. In other words, vaccines are mostly efficacious since they prevent a new (at least to the immune system) HPV strain from infecting the host. Therefore, one may argue that vaccines do not prevent recurrence, but do prevent second primary tumors .For OPSCC, there are no data available on the effect of prophylactic vaccines in patients with HPV-related disease. New inoculation or auto-inoculation is also possible in cancer of the oropharynx. However, the progression from inoculation to cancer development cannot be determined in OPSCC and the latency period is thought to be as long as 20 years, making studies much harder to be carried out or reproduced . On the Therapeutic vaccines differ from prophylactic vaccines, as they are designed to generate cell-mediated immunity against transformed cells , rather Another viable strategy is to use RNA vaccines to induce immune response against the E6 and E7 antigens . The intIn recurrent/metastatic therapeutic settings, treatment with check-point inhibitors has become the standard of care for oropharyngeal cancer since the results of the Keynote-048 trial were published . HoweverThere is limited evidence from Phase II trials indicating positive results in patients with recurrent/metastatic HPV-related disease who received checkpoint inhibitors (PD1/PDL1) in combination with E6/E7-therapeutic vaccines ,59 Tabl. These sNCT02128126, NCT04405349) for HPV-positive disease. The combination of E6-E7 immunization and inhibition of the PD1-PDL1 checkpoint, which is a mechanism of immune evasion in advanced cancers of the head and neck, is expected to double the natural immune system response against the cancer [Several clinical trials with E6/E7 vaccines are in progress in the recurrent/metastatic setting  have demonstrated that standard therapy with concomitant high-dose cisplatin effects a better cure rate than the less toxic cetuximab ,63. CurrSimilarly, in the NCT04369937 trial, intermediate-risk patients with HPV-related disease will undergo intensity modulated radiotherapy (IMRT) combined with Cisplatin, Pembrolizumab, and an experimental E6–E7 vaccine. The combination of checkpoint inhibitors with vaccines is of great interest, especially after the negative results of the JAVELIN 100 study, which showed no survival advantage in patients receiving Avelumab (compared to a placebo) in addition to radiation with high-dose cisplatin ,67. HoweAnother de-intensification strategy is to begin treatment with neoadjuvant chemotherapy or immunotherapy to reduce the tumor load, such as in the NECTORS trial . Some trThere are numerous emerging prospects for the beneficial use of therapeutic vaccines, as well as for targeted, molecular-based therapies for HPV+ OPSCC. Overall, the future for developing novel and effective therapeutic agents for HPV-associated head and neck tumors is promising and continued progress is critical to meet medical challenges posed by this growing epidemic.


Dual centrifugation (DC) is a new and versatile technique for the preparation of liposomes by in-vial homogenization of lipid-water mixtures. Size, size distribution, and entrapping efficiencies are strongly dependent on the lipid concentration during DC-homogenization. In this study, we investigated the detailed structure of DC-made liposomes. To do so, an assay to determine the ratio of inner to total membrane surfaces of liposomes (inaccessible surface) was developed based on either time-resolved or steady-state fluorescence spectroscopy. In addition, cryogenic electron microscopy (cryo-EM) was used to confirm the lamellarity results and learn more about liposome morphology. One striking result leads to the possibility of producing a novel type of liposome—small multilamellar vesicles (SMVs) with low PDI, sizes of the order of 100 nm, and almost completely filled with bilayers. A second particularly important finding is that VPGs can be prepared to contain open bilayer structures that will close spontaneously when, after storage, more aqueous phase is added and liposomes are formed. Through this process, a drug can effectively be entrapped immediately before application. In addition, dual centrifugation at lower lipid concentrations is found to produce predominantly unilamellar vesicles. Vesicular phospholipid gels (VPGs) are highly concentrated liposomal formulations that can be prepared from concentrated aqueous phospholipid mixtures by high-pressure homogenization (HPH) or dual centrifugation (DC). VPGs can be used as versatile and well-tolerated depot formulations, as reviewed by Tian et al. . On the Dual centrifugation (DC) is a new technique to rapidly prepare VPGs which can be converted to conventional liposomal dispersions by dilution as needed ,4. Dual The DC-homogenization of concentrated phospholipid dispersions to obtain liposomes is usually performed in small and disposable vials, such as 2 mL polypropylene twist-top vials, and supported by the addition of ceramic beads. For DC, the oblong sample vials are positioned horizontally in the dual centrifuge instead of the usual vertical positioning. During DC, the sample vial is centrifuged at high speed and simultaneously turned around a second rotation axis. This additional rotation in combination with its horizontal positioning causes a rapid change in the direction of the high centrifugal acceleration inside the turning sample vial. The resulting powerful movement of the viscous phospholipid dispersions, including the beads moving from the top to bottom of the vial and vice versa, results in strong shear forces and consequently the homogenization of the dispersions. Thus, DC is also used for nanomilling or tissue disruption ,7.Dual centrifugation has many advantages in the production of VPGs and the corresponding liposomes. The process is carried out in closed disposable vials , making it very simple and safe, since contact with the product is avoided and cleaning of the device is not necessary. The use of sterile vials allows for the easy preparation of sterile VPGs/liposomes. At the same time, DC-homogenization is gentler compared to high-pressure homogenization, since the energy input is lower than in HPH, but the number of homogenization events is much higher . In addiSince the parallel processing of up to 40 different formulations in very small batches is possible, DC is an attractive tool for screening for improved lipid compositions and manufacturing conditions. In a previous work , the DC-A liposome screening diagram can be divided into three typical sections regarding the lipid concentrations used for liposome preparation: In the first section, up to about 20% to 30% lipid concentration, the liposome sizes decrease, and the EE-values increase with the increasing lipid concentration. In the following section, from about 20% to 30% up to 60% to 70% lipid concentration, the liposome sizes are at their minima and fairly constant, and only the PDI-values decrease. The EE-values in this section are high and also quite constant. In the third section, starting at lipid concentrations of >60% to 70%, the EE-values drop, and the liposome sizes and PDI-values increase [Since with increasing lipid concentrations of >20% to 30%, the lipid content is higher than necessary to completely fill the volume of the resulting VPG with unilamellar vesicles, we expect the formation of multilamellar vesicles. Furthermore, at even higher lipid concentrations of >60% to 70%, the amount of water becomes too low to fully hydrate the phospholipid head groups and fill the spaces between and within liposomes ,16 to as2000 (PEG); ≥98%) were kindly provided by Lipoid GmbH . 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- 18:0 NBD-DSPE  was obtained from Avanti Polar Lipids .Hydrogenated egg phosphatidylcholine , 1,2-dipalmitoyl-sn-glycero-3-phosphocholine , 1,2-distearoyl-sn-glycero-3-phosphocholine , and N-(Carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt  was obtained from Merck . TRIS (hydroxymethyl)aminomethane  and ethylenediaminetetraacetic acid  were obtained from Carl Roth GmbH .Calcein (product number: C0875) and cholesterol (≥99%) were obtained from Sigma-Aldrich . Sodium dithionite  were prepared by dissolving the lipids in chloroform to produce a solution of 200 mM total lipid. Aliquots were pipetted into 2 mL conical screw cap vials , and the solvent was removed in a vacuum centrifuge  equipped with a vacuum pump  at 45 °C for 2–4 h. To ensure the complete removal of the solvent, the lipid films were transferred to a vacuum desiccator equipped with a high-vacuum pump  for at least 12 h.® Type ZY-P Pharma, Sigmund Lindner GmbH, Warmensteinach, Germany) and the necessary amount of aqueous phase were added to the vials already containing the lipids. For samples with NBD-DSPE, a TRIS buffer with pH 7.4  was used. TRIS buffer  was used for the cholesterol-containing samples. For the cholesterol-free samples, 25 mM NaCl was added to the TRIS buffer for osmotic balancing of the 10 mM sodium dithionite. For the samples with calcein, a TRIS buffer  with a pH of 8.5 was used. The desired lipid concentration (10–80%) was achieved by mixing the 10–80 mg lipid mixture with the corresponding 90–20 µL of aqueous buffer, respectively. The lipid concentration (%) is given as w/v in the VPG. For convenience, we approximated that 1 mg lipid per 100 µL dispersion represents 1% lipid concentration, due to a density close to 1 g/mL for both the buffer and phospholipids. Liposome preparation was performed using a dual centrifuge ) equipped with adapters for processing 40 vials in parallel . The adjustment of the temperature of the dual centrifuge concerns the rotor and the surrounding air. Due to the strong homogenization forces, the sample typically heats up by approx. 20 °C compared to the temperature set on the instrument. The resulting VPGs were typically diluted to a liposomal dispersion with TRIS buffer pH 7.4 (10 mM TRIS and 0.5 mM EDTA) for cholesterol-containing samples or an additional 25 mM NaCl for cholesterol-free samples, in the ratio 3:1 (v/v) by DC  for 2 min. For all lipid mixtures, a batch size of 100 mg vesicular phospholipid gel (VPG) was prepared to standardize the influence of the beads and wetting of the vial surface.Liposome samples were prepared as previously described . In brieThe lipid components (dry powder form) were individually weighted in the vial. The beads and buffer were added according to Dynamic light scattering  was used to determine the intensity-weighted hydrodynamic diameter (Z-average) and polydispersity index (PDI) of the DC-produced liposomes. Liposomal dispersions were diluted with TRIS buffer . The attenuator was set automatically to reach a count rate of about 150–250 kcps. The instrument evaluated the backscattering intensity from a 4 mL polystyrene cuvette with a layer thickness of 10 mm  at an angle of 173°. Three measurements with an automatically selected number of scans  were taken for each sample.VPGs containing a nitrobenzoxadiazole (NBD)-labeled lipid were diluted to a lipid concentration of 0.05 mM with TRIS buffer pH 7.4 using DC . Samples with quencher contained 10 mM sodium dithionite. Samples were prepared from a 1 M sodium dithionite stock containing 1 M TRIS; the sodium dithionite stock was prepared fresh on the same day as the quenching experiments due to the instability of sodium dithionite in an aqueous solution. The liposomal dispersion was added to the sodium dithionite-containing buffer 10 min before measurement and all measurements were conducted in an anti-photobleaching setup, whenever possible. Three different measurement methods, described below, were used to determine the loss in fluorescence intensity caused by the reduction of the NBD-headgroup.Time-resolved fluorescence (TRF) measurements were performed using the fluorescence lifetime spectrometer FluoTime 100 . The excitation source was a modular laser diode (LDH-P-C-470) with a wavelength of 470 nm (±10 nm), pulsed at a repetition rate of 5 MHz. The laser was operated with the laser driver PDL 800-D . Decay curves were recorded at a 515 nm wavelength by using time-correlated single-photon counting (TCSPC). For each sample, the measurement conditions were set to a peak count of at least 10,000 photons for a sample without a quencher. The measuring period was set to 120 s and the measurement conditions were kept the same for the samples with and without the added quencher. Fluorescence decay curves were fitted by FluoFit software  by reconvolution of a biexponential fitting function with the instrument response curve recorded using a Ludox scattering standard. IAS was calculated using total counts according to Formula (2) , the totSteady-state fluorescence measurements were conducted using a PerkinElmer LS55 fluorescence spectrometer . A spectral bandpass of 460 nm (±10 nm slit) and 535 nm (±10 nm slit) were used for excitation and emission, respectively. The fluorescence intensity at 25 °C was measured for samples with and without sodium dithionite.Steady-state fluorescence measurements in a black 96-well microtiter plate were conducted using the BioTek PLx800 fluorescence reader . The measurements were each performed in triplicate with a total volume of 200 µL per vial. A 485 nm excitation filter and a 528 nm emission filter were used. Calcein-free VPGs were prepared by DC-homogenization  of the respective lipid films and the respective volume of TRIS buffer . In the next step, 60 mM calcein buffer was added to the VPG and homogeneously dispersed by DC for HEPC/Chol 55/45 up to a total lipid concentration of 40% with 1000 rpm, for 2 min, and at 20 °C; for HEPC/Chol/PEG 50/45/5 up to 20% for VPGs with a lipid concentration lower than 55%; and up to 40% for VPGs with a lipid concentration of 55% and higher with 600 rpm, for 2 min, and at 20 °C. In the third step, the calcein/VPG mixture was diluted with TRIS buffer by DC .EE determination as previously described [After preparation, the calcein-containing liposome dispersions were diluted in the ratio of 1:5000 prior to the escribed . Time-reescribed . In brieThe samples for the cryo-EM analysis were prepared by DC as described in Previous investigations showed that important characteristics of liposomes, such as entrapping efficiencies for water-soluble compounds, size, and size heterogeneity, can be modulated through a conscious choice of the lipid concentration used during the DC-homogenization . To furtTo determine the inaccessible surface (IAS) of DC-prepared liposomes, 0.1 mol% of the fluorescence-labeled lipid NBD-DSPE was homogeneously dispersed in the lipid mixture used for liposome preparation. The fluorescence signal of NBD-DSPE at the outer (accessible) surface of the liposomes was quenched by adding the essentially membrane-impermeable sodium dithionite, which reduces the nitro-group of NBD to an amino-group A as descWith increasing IAS, the liposomes consisted of a higher number of lamellae, i.e., the lamellarity increases. Calculating the exact number of lamellae was not possible due to several reasons, such as non-homogeneous size distribution, the occurrence of intravesicular vesicles, and a decrease in the diameter with each additional lamella.The fluorescence intensities used for IAS measurements were determined with three different types of fluorescence spectrometers and compared with each other. The three instruments (averaged time-resolved fluorescence measurement (FT100), steady-state fluorescence spectrometer using cuvettes (LS55), and MTP-fluorescence photometer (PLx800)) resulted in slightly different absolute results, but the trend for AS and IAS were the same and thus sufficient to characterize the lamellarity of liposomes B.value increases from 10% to 30% lipid concentration, reaches a plateau area from 30% to about 50%, and decreases from 50% to 80% lipid concentration. The liposomes tend to be larger and more polydisperse in size when prepared from lipid/buffer mixtures with <30% or >70% lipid concentration  into the membranes of HEPC/Chol-liposomes led to clear differences in the trend for the IAS with increasing lipid concentration  surfaces of liposomal membranes by shift-reagents such as Pr3+ or Eu3+, lamellarity (L) was simply calculated by the equation: L = 100/ or by L = (peak area of both peaks)/(peak area of shifted peak) [The method used here to characterize the lamellarity of liposomal vesicles was adapted from Gruber et al.  and is bed peak) . HoweverSince it was found that IAS cannot only be determined using the very sensitive, single photon counting FT100 but also with normal steady-state fluorometers such as MTP- or cuvette-fluorescence readers using the identical liposomal dispersion, the assay supplements the already described liposomal formulation screening process  in a verThe DC-homogenization of moderately concentrated lipid/buffer mixtures  resulted in rather small, as well as unilamellar vesicles, as shown by the lamellarity assay and cryo-EM. Thus, it can be concluded that the formation of unilamellar vesicles within a VPG is preferred over multilamellar vesicles, probably driven partly by the high energy demand of forcing the rather rigid HEPC/Chol-membranes into structures with high curvature—as it is needed for the formation of small multilamellar vesicles. If excess water is available, unilamellar liposomes would be preferred over multilamellar liposomes for entropic reasons as well.Reaching about 30% lipid concentration, the steep increase in the EE-values transformed into a flatter increase , which cAt lipid concentrations higher than about 30%, there was more lipid available than could be accommodated as unilamellar vesicles in a certain volume. In an earlier study , it was Increasing the lipid content during VPG production to 30% and higher resulted in liposomes of similar hydrodynamic size but with more lamellae. Thus, since the vesicles within the VPGs produced at concentrations higher than about 30% were round and had sizes similar to the vesicles that resulted from lower lipid concentrations, we concluded that the excess of lipid at higher lipid concentrations was probably located inside the vesicles, resulting in small multilamellar vesicles. This conclusion was supported by cryo-EM pictures, as well as by the lamellarity assay which showed an increasing inaccessible surface (IAS) with increasing lipid concentrations. Our findings agree with the findings of Tardi et al. who showed freeze-fracture electron microscope pictures of vesicles prepared by high-pressure homogenization that clearly display the expected multilamellar vesicular structures .As shown by the IAS-assay and cryo-EM pictures, at a concentration of about 60%, the maximal lamellarity was reached, meaning that the vesicles were completely filled with membranes B, 60% whLiposomes are typically classified by the number and arrangement of bilayers entrapping an aqueous core. For multilamellar liposomes, a distinction between multilamellar (MLVs) and multivesicular vesicles (MVVs) was made. For unilamellar vesicles, three classifications according to their size are common: giant unilamellar vesicles , large unilamellar vesicles , and small unilamellar vesicles  . MLVs anDue to their higher membrane content, SMVs are also promising carriers for lipophilic or amphiphilic drugs. The higher vesicle stiffness caused by the additional lamellae of SMVs seems to improve their cellular uptake . This inThe expected slow release of water-soluble drug compounds makes SMVs also promising drug carriers within depot formulations, for intramuscular, intraperitoneal, or subcutaneous applications. Due to the fact that those depot formulations have to be injected by syringes using thin needles, the currently used VPG-based depot-formulations are made at rather low lipid concentrations ,31. ThisBased on the idea that it is mainly the lipid concentration used for VPG preparation that determines the formation of unilamellar vesicles or SMVs, it is conceivable that other techniques such as HPH which are basically suitable to prepare VPGs will result in comparable vesicles after dilution of the VPGs. One disadvantage of HPH in contrast to DC is that the resulting VPGs usually have to be autoclaved prior to use which results in an unwanted increase in the size of the final vesicles . Due to At very high lipid concentrations such as >65% for HEPC/Chol-mixtures, DC-homogenization results in large and heterogenous vesicles with decreasing EE-values. Let us estimate the minimal amount of water needed to maintain closed multilamellar structures: Approximately 12 water molecules are necessary to hydrate a phospholipid headgroup ,34,35 whClosed vesicular structures, however, need additional water to fill the interstices between the vesicular structures and their inner core. For densely packed, equally sized spheres, the theoretical fraction of interstitial space is 26 v%. This fraction is reduced for polydisperse, deformable bodies but for interstices plus cores, we may still estimate a space requirement of 10–30 v%. Adding this to the 22% PC-bound water (s. above) results in a minimal amount of water of about 32–52% (or a maximum lipid concentration of 48–68%) which is necessary to form a DPPC-VPG consisting of closed vesicular structures. Since the vesicles within a typical VPG are typically heterogeneous in size and lamellarity, one must expect the appearance of open stacks of lamellae coexisting of closed structures in VPGs of 48–68% lipid concentration for pure DPPC-VPGs. Irrespective of the heterogeneity of the vesicles, at about 70% phospholipid, no closed structures are expected to exist anymore.However, the HEPC/Chol-mixture used in this study to produce VPGs consisted of only 71.4% phospholipid and 28.6% cholesterol, which binds less water than phospholipids, meaning (i) that the formation of open lipid stacks will start at somewhat higher lipid concentrations and (ii) that closed vesicles are also expected at somewhat higher lipid concentrations. The experimental finding that the amount of open lipid stacks is maximized at 78% lipid concentration for HEPC/Chol-VPGs is therefore in good agreement with the above-made hypothesis.Diluting calcein-free VPGs with a calcein solution resulted in an increased entrapment of the added dye , startinHowever, the low uptake of calcein noted at lower lipid concentrations can be interpreted as an artifact of the EE-calculation routine . SpecifiCryo-EM pictures of VGPs made from 80% HEPC/Chol confirmed the existence of large multilamellar vesicles but also showed numerous vesicle-in-vesicle structures. The fact that no open membrane stacks and only closed structures are visible in the cryo-EM-pictures can be explained by the dilution of the highly concentrated VPGs that was necessary to prepare the samples for cryo-EM. The re-closed open stack structures are the result of a lack of water during the DC-homogenization process. Following this assumption, no homogenous liposomes can be formed anymore, and only large non-homogeneous vesicles are formed. This can be observed by the sharp increase in the size and the size distribution (increasing PDI) of the vesicles prepared by DC-homogenization between 70% and 80% lipid concentration A.2000. The curvature-active PEG lipid may also stabilize the dehydration-induced open lamellar stacks by covering their open edges—at a low PEG-lipid content, they would redistribute over the membrane as the sheets re-close upon dilution.In accordance with the fact that PEG chains bind additional water molecules, the uptake of calcein in open lipid structures started at somewhat lower lipid concentrations (about 50%) when using a lipid-mixture containing 5 mol% DSPE-PEGIn the case of HEPC/Chol liposome dispersions prepared by conventional methods, it has previously been shown by cryo-EM that the inclusion of 5 mol% PEG-lipid induces the formation of a small population of lipodisks . The lipThe newly developed in-vial homogenization using dual centrifugation (DC) is by far the easiest and fastest way to prepare liposomes from various lipid compositions, but it is not fully understood in detail. Here, we identified two novel liposomal preparations with considerable potential for pharmaceutical application. Further, in addition to largely unilamellar vesicles, we were able to produce small multilamellar vesicles (SMVs) as well as self-entrapping open bilayer stacks that can be stored and then used to spontaneously entrap an aqueous solution of a drug that is being added before administration.In more detail, the investigation of the lamellarity of DC-made liposomes using cryo-EM and a fluorescence-based lamellarity assay substantially improved our understanding of the vesicle-forming processes and the morphology of the resulting liposomes. The insights gained concerning the structural behavior of DC homogenized lipid mixtures composed of HEPC/Chol 55/45 are summarized in Further increasing the lipid concentration to 40%—which is more lipid than can be accommodated in densely packed, unilamellar liposomes—had only a minor influence on the already small vesicle size, however, multilamellar and multivesicular vesicles then became dominant. At about 60% lipid concentration, the lamellarity was at its maximum. Cryo-EM pictures show vesicles almost completely filled with membranes that are very closely packed. The very narrow size distributions are probably due to the higher stability of the multilamellar vesicles against shear stress during homogenization.At about 65% lipid, there is not enough water to fully hydrate the phospholipid headgroups and fill interstices between and the remaining cores within vesicles. With increasing lipid content in the VPG, closed vesicular structures are increasingly replaced by open lamellar stacks that re-close and, in this way, entrap the calcein solution added upon dilution. These re-formed vesicles are rather big and heterogeneous. At about 80% lipid, the remaining 20% water is almost completely needed as lipid-bound, interlamellar water, and virtually no closed structures are left in the gel.Adding PEG-containing lipids to the lipid mixture caused the formation of open membrane sheets already at medium lipid concentrations, an effect which was likely due to the high water-binding capacity of PEG and the stabilization of the edges of the open membrane sheets by PEG.In addition to providing a better understanding of the processes during liposome preparation using DC, the new findings allow for the specific tailoring of unilamellar as well as small multilamellar vesicles (SMVs) with encouraging entrapping efficiencies for water-soluble as well as lipophilic compounds by simply adjusting the lipid concentration during DC.


The aim of the nationwide prospective seroconversion (PROSECO) study was to investigate the dynamics of anti-SARS-CoV-2 IgG antibodies in the Czech population. Here we report on baseline prevalence from that study.The study included the first 30,054 persons who provided a blood sample between October 2020 and March 2021. Seroprevalence was compared between calendar periods, previous RT-PCR results and other factors.The data show a large increase in seropositivity over time, from 28% in October/November 2020 to 43% in December 2020/January 2021 to 51% in February/March 2021. These trends were consistent with government data on cumulative viral antigenic prevalence in the population captured by PCR testing – although the seroprevalence rates established in this study were considerably higher. There were only minor differences in seropositivity between sexes, age groups and BMI categories, and results were similar between test providing laboratories. Seropositivity was substantially higher among persons with history of symptoms (76% vs. 34%). At least one third of all seropositive participants had no history of symptoms, and 28% of participants with antibodies against SARS-CoV-2 never underwent PCR testing.Our data confirm the rapidly increasing prevalence in the Czech population during the rising pandemic wave prior to the beginning of vaccination. The difference between our results on seroprevalence and PCR testing suggests that antibody response provides a better marker of past infection than the routine testing program. Piler et al. perform a longitudinal serology study of the Czech population between October 2020 and March 2021 and describe an increase in anti-SARS-CoV-2 IgG antibodies that correlates with SARS-CoV-2 PCR testing data. They include data stratified by age, sex, body mass and history of symptoms. The nationwide prospective seroconversion (PROSECO) study is monitoring the presence of anti-SARS-CoV-2 IgG antibodies in the Czech population. The presence of these antibodies is indicative of a previous infection with SARS-CoV-2. Here, we report initial results from 30,054 participants in this study that were recruited between October 2020 and March 2021, during the second wave of the COVID pandemic. The data indicates that the percentage of the population infected with SARS-CoV-2 is higher than estimates based on official data on cumulative PCR testing for presence of the virus. At least one third of participants with antibodies did not have symptoms of COVID and 28% had not undergone PCR testing. This study demonstrates the importance of monitoring the presence of anti-SARS-CoV-2 IgG antibodies to accurately assess the proportion of the population that has been infected over time. This virus stimulates a rapid seroconversion to IgG antibodies in symptomatic3 as well as asymptomatic subjects5. Therefore, the memory IgG antibodies against SARS-CoV-2 can serve as a specific long-term biomarker of previous SARS-CoV-2 infection6 as well as an appropriate marker for monitoring the persistence of antibodies against SARS-CoV-2 after vaccination7.The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)8, shows a lack of nationwide population-based seroprevalence studies in Central and Eastern Europe, compared to several seroprevalence studies in other parts of Europe11. While some existing studies have examined general population samples , most have been restricted to hotspots17. These studies adopted different sampling and analytical strategies: some have used household and community sampling17 while others focused on biosamples from blood donors18 or residual sera in laboratories12. While the majority of these studies employed serological tests, point-of-care antibody assays with lower sensitivity and specificity were also used19. A review by Rostami et al. reported seroprevalence for Europe between 0.66% and 5.27% until August 202020. Thus far, only a small number of nationwide population-based studies have been published covering the period between November 2020 and March 2021, i.e. the second wave of the epidemic in Europe23.The SeroTracker dashboard, which systematically monitors and synthesises findings from hundreds of global SARS-CoV-2 serological studies24.In the Czech Republic, the first COVID-19 cases were confirmed on 1 March 2020. The first epidemic wave in spring 2020 was relatively modest, with only 9301 cases recorded by the end of May 2020 . The governmental restrictions were dismantled in the summer; this resulted in a worsening of the epidemic situation in autumn 2020. At the peak of the second wave, over 10,000 new cases were diagnosed every day  and the Czech Republic ranked among the countries with the greatest burden of COVID-19 in Europe and in the world. As of 31 May 2021, the Czech Republic had a cumulative total of 1,661,787 cases confirmed  and the highest cumulative incidence of COVID-19 among countries with ≥1,000,000 inhabitantsTo address the lack of regional data and to investigate the dynamics of seroprevalence of anti-SARS-CoV-2 IgG antibodies in the region, a nationwide prospective seroconversion study (PROSECO) was initiated in the Czech Republic. The aim was to study the dynamics of seroconversion in three six-month-long periods: (1) the second epidemic wave (unvaccinated population); (2) the mass vaccination period; and (3) the post-vaccination period. Here, we describe the study design and the results of the first phase of the study (October 2020 to March 2021). Our findings show a rapid increase in seroprevalence in the Czech population during the rising pandemic wave prior to the beginning of vaccination and suggest that antibody response provides a better marker of past infection than the routine testing program.The Czech PROSECO study investigates seroconversion after SARS-CoV-2 infection (or vaccination), as well as the decline of memory IgG antibodies against SARS-CoV-2 over time. The study has been planned as a series of three consecutive phases, each lasting six months, with participant enrolment taking place in the first phase. The first phase of the study, described here, was completed just before the launch of a massive nationwide vaccination programme.All clients of the second-largest health insurance company in the Czech Republic were sent a written invitation to participate in the study. On September 20 the first batch of invitation letters was sent to all clients (>18 years old) and the first 30,054 persons who provided blood samples between 1 October 2020 and 31 March 2021 were included in the study population. The presence of COVID-19 symptoms on the day of study enrolment was used as the sole exclusion criterion. The participants paid <20% (i.e. a negligible sum) of an antibody test cost.The participants were invited to visit their local blood collection centres managed by one of the five different chains of clinical laboratories incorporated to QualityLab association covering the entire area of the Czech Republic. A 3 ml blood sample was collected in a separating gel vacuum tube. Serum samples were distributed at +4 °C and subsequently analysed in central laboratories within 12 h. Leftover sera were stored in a biobank for future use.Participants completed a questionnaire that included personal information , anthropometric data, self-reported results of previous RT-PCR tests (if performed), history of symptoms compatible with COVID-19, and records of COVID vaccination.Informed consent forms were obtained from all study participants during each wave of the data collection. An ethics committee approval of all aspects of data collection, as well as of the secondary data analysis, was obtained from the ELSPAC ethics committee under reference number (C)ELSPAC/EK/5/2021.CE-marked serological tests were performed in accredited clinical laboratories. Antigen-specific humoral immune response was analyzed by detection of IgG antibodies against the spike protein using commercial immunoassays LIAISON SARS-CoV-2 S1/S2 IgG  and SARS-CoV-2 IgG II Quant . Testing was conducted on the LIAISON XL  and on the Alinity  respectively. Samples were tested individually and reported according to the manufactures´ criteria.25. We used a multivariate Poisson regression model with a robust error variance to evaluate the differences in the seroprevalence rate between study periods, participant characteristics and testing providers and to compare SARS-CoV-2 antibody reactive individuals to non-reactive individuals. Differences in prevalence were expressed as prevalence rate ratios (PRRs) between each category and reference group. We used standard descriptive statistics to characterize the study data set.The primary aim of this study was to determine the seroprevalence of the adult Czech population. We estimated seroprevalence rates and 95% confidence intervals. We also standardized the seroprevalence rates to age and sex, using the Czech population as a standardPopulation data on COVID-19 were obtained from the Czech Central Information System of Infectious Diseases (ISID), which includes records of all consecutive patients with COVID-19 in the Czech Republic identified and confirmed by laboratory testing. ISID data are routinely collected in compliance with Act No. 258/2000 Coll. On the Protection of Public Health and are publicly available in aggregated and anonymized form of open or authenticated data sets. All analyses were conducted using Stata version 15.1 .Between October 2020 and March 2021, 30,054 individuals consented to participate in the study and their results were included in the final analyses. The seroprevalence overview is shown in Table p-values p < 0.001). A higher seropositivity prevalence was also observed in persons with BMI > 30 kg/m2 (Supplementary Data p = 0.086) and participating laboratories .Table Table Figure This study demonstrated a rapid increase in anti-SARS-CoV-2 IgG antibodies in the unvaccinated Czech adult population during the second COVID-19 pandemic wave between October 2020 and March 2021. We found a massive increase in seroprevalence of IgG antibodies against SARS-CoV-2 from 9 to 51.0% over the course of six months , which is in agreement with similar studies performed in other countries until September 2020. Data available for a similar period from the Netherlands, France, Spain, UK, and Italy, for instance, suggested seroprevalence between 3.4 and 11.6%26, no difference was found between men and women . Similar to previously published studies such as Ward et al. in the UK13, we also established a higher risk for the obese sub-population. These findings can be partially explained by sociological data from a national survey monitoring behaviour of various population groups and their adherence to the restrictions during the pandemic (zivotbehempandemie.cz).Only small differences in the seroprevalence of anti-SARS-CoV-2 IgG antibodies were observed among various population groups. Similar to the previous studies13. The ratio of asymptomatic and symptomatic participants differs among study phases: twice as many seropositive participants were asymptomatic in the third vs. in the first phase. This could be explained by exposure to low doses of the virus repeatedly over time, which could lead to seroconversion without the manifestation of symptoms  of the spike protein of SARS-CoV-2 virus. Since previous studies have shown that the SARS-CoV-2 spike protein RBD is the target of neutralizing antibodies, this molecule has become a particularly interesting target for serological testing and vaccine development32. Our study also has the advantage of including data from serological testing as well as self-reported RT-PCR test results. This allows us to explore the spread of infection more precisely in the nationwide study population.To the best of our knowledge, the Czech PROSECO study is the largest study of the seroprevalence of anti-SARS-CoV-2 IgG antibodies in Central and Eastern Europe. According to the WHO seroepidemiological investigation protocolThe study also has important limitations. The main weakness is the self-selection of study participants. Potential participants were contacted through their health insurance company  and using online advertising. Similar to other national seroprevalence studies, children were excluded for obvious reasons . The first 30,054 volunteers reporting to the QualityLab network were then enroled in the study, after which the recruitment procedure was discontinued. As a result, the true response rate cannot be determined and, despite its large size, this study sample may not reliably represent the entire population, since the participants were self-selected volunteers rather than a random sample selected from a specific sampling frame. It is likely that persons at higher risk of past or current infection are overrepresented in this study, which would lead to an overestimation of the absolute seropositivity prevalence rate. As we currently do not have information about socio-economic status and geographical location of study participants, we were unable to further assess the potential selection bias and to evaluate geographical differences in seropositivity. Crucially, however, the temporal trend in seropositivity in this study sample closely reflects the cumulative PCR positivity trend in national data, thus suggesting the good internal validity of our results.Compared to the national statistics, female and middle-aged participants (40–59 years) were over-represented in our study at the expense of males, respondents from 18 to 39 years, and those over 60 years of age. The discrepancy in age profile can be partially attributed to the fact that the study participants were clients of one particular health insurance company, which has a larger representation of middle-aged clients compared to the general population. We also excluded institutionalised persons, which resulted in an underestimation of the oldest age category living in retirement/nursing homes, and individuals who were in prisons or monasteries at the time of the study. Availability of socio-economic and geographical data in future phases of the study will allow us to better interpret the findings and assess regional trends.33.In March 2021, the national vaccination program started, aiming at vaccinating the entire population against SARS-CoV-2 by the end of September 2021. The second phase of the PROSECO study was therefore launched to follow-up on the study participants throughout the most intense vaccination period (i.e. from April to September 2021). The third phase, planned from October 2021 to March 2022, will investigate the dynamics and persistence of IgG SARS-CoV-2 antibodies. This longitudinal follow-up, which thus includes pre-vaccination, vaccination and postvaccination periods, together with the potential linkage of resulting data to morbidity and mortality data from the national registers will further increase the importance and impact of this surveillanceIn conclusion, the results of the first phase of the PROSECO study indicate that a significant part of the Czech adult population is no longer immunologically naïve to the SARS-CoV-2 infection. The massive increase of seroprevalence demonstrates the extensive exposure of the Czech population to the SARS-CoV-2 virus between October 2020 and March 2021. The number of seropositive participants who never underwent RT-PCR testing demonstrates the importance of serological population-based studies describing the spread and exposure to the virus in the population over time. Seroconversion and positivity in specific antibody testing is a powerful additional diagnostic surrogate marker of SARS-CoV-2 antigenic challenge.Description of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Description of Additional Supplementary FilesPeer Review File


The analysis of the effectiveness of booster shots compared with primary vaccination is extremely vital. This paper aimed to summarize the results of all available evidence studies on the effectiveness of booster vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Articles published up to 21 June 2022 were systematically searched through PubMed and EMBASE databases. The searched studies were independently assessed for quality using the Newcastle–Ottawa Scale. Results: Seven studies (nine datasets) met the criteria and were included in this study. The pooled results demonstrated a 71%  reduction in SARS-CoV-2 infection rates among subjects who received a booster shot compared with those who did not receive a booster shot of coronavirus disease (COVID-19) vaccine. In addition, this analysis emphasized that during the period when the Delta variant was predominant, subjects who received the booster shot showed an 82%  reduction in infection rates. Moreover, during the period of dominance of the Omicron variant, subjects who received the booster vaccination displayed a 47%  reduction in infection rates. This finding confirmed that booster vaccination against the Omicron variant is significantly less effective than that against the Delta variant. In pandemic periods, correlations between the dominant variant and the efficacy of the COVID-19 vaccine booster should be considered when making vaccine booster plans. Coronavirus disease COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization (WHO) on 11 March 2020 [9, which Although COVID-19 vaccination is fully implemented worldwide, SARS-CoV-2 remains rampant. As with other vaccines, the protective efficacy of the COVID-19 vaccine weakens over time after the completion of vaccination. A meta-analysis ,4. ThereThe most notable feature of the epidemic is the constant and rapid mutation of SARS-CoV-2. As of 13 August 2022, WHO has identified five variants of concern (VOCs), namely Alpha, identified on 18 December 2020; Beta, identified on 18 December 2020; Gamma, identified on 11 January 2021; Delta, identified on 11 May 2021; and Omicron, identified on 26 November 2021 . CurrentThe effectiveness of a third dose (booster) compared with the two doses in a real-world setting is unknown, and a meta-analysis of the effectiveness of real-world-based booster shots compared with primary vaccination is extremely important. Concerns regarding the possible lower vaccine effectiveness against the Omicron variant compared with that against the Delta variant have emerged. Whether a difference in vaccine effectiveness between the booster doses during the Omicron- and Delta-dominant period should be determined to address both questions. Therefore, this paper aimed to conduct a meta-analysis to summarize the results of all available evidence and studies on the effectiveness of booster vaccination compared with primary vaccination against SARS-CoV-2.This meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. This study was prospectively registered on PROSPERO (n.CRD42022337356). Articles published up to 21 June 2022 were systematically searched through PubMed and EMBASE databases. The search keywords included (“SARS-CoV-2” or “COVID-19” or “2019-nCov”) AND (“Vaccine” or “Vaccination”) AND  AND (“Efficacy” or “Effectiveness”).The inclusion criteria included (1) studies with outcomes of COVID-19 infection; (2) cases defined as individuals who tested positive for SARS-CoV-2 as confirmed by polymerase chain reaction, (3) observational studies, (4) studies published in English; (5) studies that excluded patients who had been infected with COVID-19 prior to their first vaccination.The exclusion criteria included (1) articles for which the full text could not be found; (2) systematic reviews, commentaries, case reports, letters, and guidelines; (3) studies of non-human subjects, (4) articles for which data could not be extracted; (5) studies considering only hospitalization, serious illness, death and other serious outcomes without considering SARS-CoV-2 infection.Data extraction and quality assessment were carried out independently by two authors, with disagreements identified by a third author. The following data were extracted from the included articles: name of first author, country, study design, vaccine, dominant variant, age range, and the number of booster and non-booster vaccinations in cases and controls. The included studies were independently assessed for quality using the Newcastle–Ottawa Scale (NOS), which was designed for observational and non-randomized studies. Scores of 0–3, 4–6, and over 7 stars were considered of low, moderate, and high quality, respectively.I2 statistic was applied to assess the heterogeneity between studies. If I2 > 50%, which indicates a high heterogeneity, the random-effects model was recommended. Meanwhile, if I2 < 50%, the fixed effects model was normally employed for the analysis. Pooled odds ratio (OR) and 95% confidence interval (95% CI) estimates were calculated to determine the association between COVID-19 vaccine booster shots and infection. Dominant variants were considered for subgroup analysis to further identify the sources of heterogeneity. A two-tailed p < 0.05 was deemed to be statistically significant. Statistical analyses were conducted by STATA/SE version 16.0 .The A total of 904 records were identified from the literature search. After removing duplicates, 578 articles were identified and subsequently screened by title, abstract, and full text. After excluding the ineligible studies from the title or abstract screening, 35 studies were reviewed in full text. As a result, seven studies (nine datasets) met the criteria and were included in this study. Studies reporting the efficacy of different dominant variants were treated as a separate dataset in the meta-analysis .n = 7). Most of the included research (n = 4) was conducted in the United States. The meta-analysis included 5,510,606 subjects, of whom 867,361 were infected patients. Of the seven studies, one was a retrospective cohort research study, and the rest were test-negative design studies. All of these studies were published in 2022, and most were conducted on mRNA vaccines. In addition, the quality of all included studies was assessed with the NOS. Four and three studies showed moderate and high quality, respectively.I2 > 50%), the correlation between COVID-19 vaccine booster shots and infections was analyzed using a random-effects model. The pooled results revealed that the COVID-19 vaccine booster was a protective factor against infection relative to the non-booster doses . A subgroup analysis was subsequently conducted based on the dominant variant strains: the Delta- and Omicron-dominant phase groups (p < 0.001). During the dominance period of the Delta variant, the booster-vaccinated subjects demonstrated a significant reduction in infection rates compared with non-booster-vaccinated subjects . During the period of dominance of the Omicron variant, booster-vaccinated subjects also displayed a reduction in infection rates compared with those who did not receive the booster vaccine . This finding supported the effect of different variant strains on the effectiveness of booster vaccination.Based on the degree of heterogeneity of the included studies (e groups . This ann < 10). Sensitivity analysis by excluding one study at a time had no significant effect on the results . This result confirmed that booster vaccination against the Omicron variant was significantly less effective than that against the Delta variant. Compared with the Delta variant, the Omicron variant has a larger area of variation, and its “face” is largely different and more infectious than the original strain [This meta-analysis is the first to report the pooled data on the effectiveness of the COVID-19 vaccine booster shot in comparison with the non-booster shot. Of the included studies, apart from one research study that did not specify an age range, all covered young people and older people aged 18 years and over. Furthermore, the sample sizes of the individual studies ranged from several thousands to several millions. One study was retrospective cohort research, and the other six applied a test-negative design, which is commonly used in vaccine efficacy studies. The results of the studies included in the meta-analysis were consistent. The pooled results demonstrated a 71%  reduction in SARS-CoV-2 infection rates among subjects who received a booster shot compared with those who did not receive a booster shot of COVID-19 vaccine. Moreover, this analysis emphasized that during the period when the Delta variant was predominant, subjects who received the booster shot showed an 82%  reduction in infection rates. In addition, during the period of dominance of the Omicron variant, subjects who received the booster vaccination displayed a 47%  reduction in infection rates. This finding supported the effect of the different variants on the effectiveness of booster vaccination. Booster vaccination during the Delta variant-dominant period was more effective in preventing COVID-19 disease than the booster vaccination during the dominant period of the Omicron variant . In addition, given the sparse literature, no distinction was made between different age groups, and data on the effectiveness in preventing severe infections were not available. The potential differences in effectiveness of preventing infection or severe infections between populations could not be compared. Focus on the discrepancies between homologous and heterologous reinforcements was also lacking. Also, in the absence of data, whether there were potential differences associated with the type of vaccine used for the first two doses and for the booster dose was not analyzed. Furthermore, the level of epidemiological dynamics and vaccine policies in each country could also affect the assessment of the effectiveness of booster doses. The major weakness was that the booster shot was administered later compared with the non-booster shot, and the effect of time on the effectiveness of the booster shot was not ruled out. Similarly, in comparison with the Delta strain, the Omicron strain emerged later with an accompanying tendency for the antibodies to fade away. Duration is the most critical confounding factor. Although the time confounder cannot be ruled out, public vaccination did not take place at the same time, and some people were vaccinated after the Omicron epidemic. Therefore, the time of infection with the Delta strain after vaccination and the time of infection with Omicron after vaccination were not totally incomparable. It is merely that information on time was not available due to data limitations. In the same studies included in the meta-analysis, the time of cases and controls were comparable, and hence they were somewhat comparable in the time of cases and controls across the meta. Therefore, despite the fact that time was the most critical confounding factor, the conclusion of this article could provide some suggestive points. More detailed information on the COVID-19 vaccine booster and all the potential factors that can affect the outcome are worthy of attention.The present meta-analysis had several limitations. In particular, the analysis of between-study heterogeneity and subgroup analyses failed to reduce the overall heterogeneity. The use of funnel plots and Egger’s test for publication bias assessment was not feasible on account of the small number of studies included in the pooled analysis (In summary, preliminary data on the COVID-19 vaccine booster suggested that potential dominant strain differences occur in terms of the efficacy. In pandemic periods, correlations between the dominant variant and the efficacy of the COVID-19 vaccine booster should be considered when making vaccine booster plans.This meta-analysis summarized the efficacy of the COVID-19 vaccine booster versus non-booster in reducing infection rates, and the findings supported the comparable effectiveness of the booster. In addition, the booster was less effective against the Omicron variant than the Delta variant. Further studies on the effectiveness of real-world vaccine booster shots are encouraged to explore other sources of heterogeneity that may influence the efficacy of meta-analysis results.


Since the outbreak of the COVID-19 pandemic, the disease has spread worldwide with more than 603 million confirmed cases and with a death toll surpassing 648 million as of 6 September, 2022.Early in the pandemic, dexamethasone was proposed as a potential treatment for patients with severe and critical COVID-19 as it suppresses the “cytokine storm” induced by SARS-CoV-2. Given the large number of clinical studies supporting the use of dexamethasone in COVID-19 patients, the most robust evidence came from the Randomized Evaluation of COVID-19 Therapy (RECOVERY) trial conducted by researchers at Oxford University. Low dose dexamethasone (6 mg once daily) has been proven to reduce the 28-day mortality rate for COVID-19 patients on either invasive mechanical ventilation or oxygen therapy . DespiteFor COVID-19 patients in LMICs such as Lebanon, cost and availability of treatment is very essential. Lebanon is a country facing unprecedented economic crisis that is pushing the country's healthcare system to the brink and creating disastrous medicine shortages. Therefore, for Lebanon, having an effective treatment such as dexamethasone, which is at the same time inexpensive, is highly significant.The author confirms being the sole contributor of this work and has approved it for publication.