Plasmodium falciparum infection were found to be of immunoglobulin G 1 (IgG1) and IgG3 subclasses and of high affinity to the target epitopes. These target epitopes cannot be characterized using recombinant antigens because of a lack of appropriate glycosylation, phosphorylation, methylation, and bisulfide bond formation, which determine the structure of conformational and nonlinear epitopes within the tertiary and quaternary structures of native P. falciparum antigens.Antibodies to blood stages protective against complications of P. falciparum schizont antigens, eliciting a protective immune response.This study aims to develop a method for the comprehensive detection of all P. falciparum antibody titers. Antigens eliciting the protective responses are identified as immunorelevant from the comparison of spot positions, indicating high-affinity IgG1 or IgG3 responses on the western blot, which is unique to or consistently more intensive in clinically immune individuals compared with nonimmune individuals. The results obtained are validated by using affinity chromatography.Purified parasitophorous vacuole membrane–enclosed merozoite structures (PEMSs) containing native schizont antigens are initially generated, separated by two-dimensional (2D) gel electrophoresis and blotted onto nitrocellulose. Antigens eliciting a protective antibody response are visualized by incubation with sera from patients with clinical immunity. This is followed by the elution of low-affinity antibodies with urea and detection of protective antibody responses by incubation with anti-IgG1 and anti-IgG3 antibodies, which were conjugated to horseradish peroxidase. This is followed by visualization with a color reaction. Blot signals are normalized by relating to the intensity of blot staining with a reference antibody and housekeeping antigens. Results are corrected for intensity of exposure by the relation of antibody responses to global P. falciparum. The sera of patients allowed the detection of 42 antigenic spots on the 2D immunoblot. The spots detected were excised and subjected to mass spectrometry for identification. A total of 19 protein spots were successfully identified and corresponded to 13 distinct proteins. Another group used immunoaffinity chromatography to identify antigens bound by IgGs produced by mice with enhanced immunity to Plasmodium yoelii. Immunorelevant antigens were isolated and identified by immobilizing immunoglobulin from immune mice to a Sephadex column and then passing a blood-stage antigen mixture through the column followed by the elution of specific bound antigens with sodium deoxycholate and the identification of those antigens by western blotting with specific antibodies.Another group previously applied 2D western blotting to analyze antibody responses to protein species) generated by posttranscriptional modifications such as phosphorylation, glycosylation, and methylation. The process involved in 2D western blotting enables highly sensitive detection, high resolution, and preservation of antibody responses during blotting. Validation by immunoaffinity chromatography can compensate for the antigen loss associated with the blotting process. It has the potential for indirect quantification of protective antibody responses by enabling quantification of the amount of eluted antibody bound antigens through mass spectrometry.2D western blotting using native antigens has the potential to identify antibody responses selective for specific defined isomeric forms of the same protein, including isoforms (PRR1-10.2196/15690 Plasmodium falciparum malaria resulted in a reduction of the parasite count to <1% of the initial value and a progressive reduction of clinical symptoms [. Immunoglobulin G 3 (IgG3) is the most effective subclass for activating the complement pathway. It is known to mediate cell lysis by Fc receptor–bearing monocytes or lymphocytes. This is particularly important in the context of immunity to P. falciparum. By their ability to link antigens to monocytes, these subclasses can stimulate them to produce tumor necrosis factor. This cytokine induces the production of nitric oxide, which kills intracellular parasites as part of antibody-dependent cellular inhibition. The generated nitric oxide is a vasodilator that prevents cerebral vasospasm, which is significantly involved in the pathogenesis of cerebral malaria [P. falciparum by monocytes, thereby indicating that its activity may be related to the ability to collaborate with monocytes [P. falciparum infection in malaria endemic areas [P.falciparum blood-stage antigens was made by using one-dimensional (1D) sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and immunoblotting [P. falciparum schizonts in clinically immune Senegalese adults and semi-immune Amazonian adult patients [Previously, the transfer of gamma globulins from immune Gambian adults to children with symptoms ,3. This symptoms . Immunog malaria . A shift malaria . IgG1 an malaria . Antibodonocytes -10. Thisonocytes ,11-15 anonocytes ,17. Focuonocytes ,19. A reic areas . Progresblotting . The firpatients . Aviditypatients -26. Patipatients ,28.P. falciparum has a 23-megabase genome that encodes an estimated 5268 putative proteins [Escherichia coli–visualized IgG responses in 53 Gambian asymptomatic parasitemic children, 81 children with clinical malaria, and 55 children without malaria. The analysis took into account only the presence or absence of specific antibodies to individual antigens as markers for immune priming. Antibody titers were not included. No association between clinical outcome and recognition of an individual antigen was observed [P. falciparum proteins. Generated by polymerase chain reaction (PCR)/recombination cloning, the proteins were individually expressed with >90% efficiency in E. coli cell-free in vitro transcription and translation reactions and printed directly without purification onto microarray slides. The protein microarrays were probed with human sera from 1 of 4 groups that differed in immune status: sterile immunity or no immunity against an experimental challenge following vaccination with radiation-attenuated P. falciparum sporozoites, partial immunity acquired by natural exposure, and no previous exposure to P. falciparum. A total of 72 immunoreactive proteins were identified, and 48 were particularly reactive. The average molecular weight of all P. falciparum proteins in the proteome is 87 kDa, but the 48 immunodominant antigens averaged nearly two times larger, presumably reflecting the presence of additional B-cell epitopes in the larger sequences [P. falciparum proteome, including 1204 known and hypothetical proteins, using sera from people in Mali exposed to seasonal malaria and monitoring IgG responses to these antigens before and after the malaria season. Of these, 491 antigens were found to be reactive with IgG. Of these, 25.2% (124/491) were derived from sporozoites, 5.5% (27/491) from merozoites, 16.8% (82/491) from trophozoites, 20.6% (101/491) from gametocytes, and 31.9% (157/491) from unknown origin. A total of 40% (196/491) of the immunogenic proteins were expressed in the membrane of the parasite or host erythrocytes. A comparison of antibody profiles of children who did not experience malaria (despite at least one positive blood smear) with those who experienced ≥1 malaria episode during the 8-month study period showed that in the 12 protected children, antibody levels were significantly higher than in the 29 unprotected children for 49 proteins, and in a subsequent study, 107 proteins were associated with a protective antibody response [proteins . To enlaobserved . This liequences . Followiresponse -36.E. coli), yeast, and baculovirus. The bacterial expression system has been by far the most popular choice to express helminth antigens. However, except for the results with cestodes, the levels of protection induced by E coli recombinants have been rather disappointing. E coli expressing antigens of trematodes and nematodes have had more variable successes, ranging from a 100% reduction in egg counts in mice and sheep for a Schistosoma mansoni antigen tested against Fasciola hepatica to a 0% reduction for nematode antigens of Haemonchus contortus and Ostertagia ostertagi. Yeast has been used to express 12 different antigens from 6 helminth species. The levels of protection obtained with these recombinants vary considerably, although all of them appear to induce some level of protection. The best results were obtained with the recombinant Sh28GST antigen from Schistosoma haematobium and the Ac-APR-1 antigen from the hookworm Ancylostoma caninum: vaccination with these antigens reduced the egg output by 77% and 85%, respectively. Although the yeast and baculovirus expression systems clearly have the capability to glycosylate the recombinant antigens, this glycosylation can differ drastically from the helminth glycans. The yeast Saccharomyces cerevisiae, for example, can cover the peptide core with very large glycan trees, which potentially mask important peptide epitopes or which can make the protein hyperantigenic [To generate vaccine antigens for anthelmintic vaccines, 3 different expression systems have been used, that is, bacterial  gel electrophoretic methods. This was illustrated by the example of in spots .P. falciparum is illustrated by the fact that striking visual evidence of widespread posttranslational modifications in the parasite was observed when a study used 2D electrophoresis followed by the mass spectrometric identification of proteins of P. falciparum ring stages, with 10 out of the 28 gene products identified in two or more variants of the same protein identified on the 2D gel as placed in different positions due to a different electrophoretic migration [E coli lack glycosylation The role of glycosylation was investigated by examining the specific binding of each antibody subclass to P. falciparum periodate-sensitive epitopes following treatment of antigen-coated microplate wells with sodium periodate at acidic pH. Such treatment has been shown to cleave carbohydrate vicinal hydroxyl groups without affecting polypeptide chains. The investigators found that 35% to 46% of all anti–P. falciparum IgG1 antibodies and 12% to 28% of anti–P. falciparum IgG3 naturally acquired by African subjects and Amazonian patients were shown to recognize periodate-sensitive epitopes [The limitations associated with a bacterial cell-free expression system used to generate protein microarrays are the lack of posttranslational modifications, such as phosphorylation, methylation, and glycosylation, and the absence of the complexity of protein folding and multimerization forming tertiary and quaternary structures, for example, those formed by disulfide bonds. The potential importance of posttranslational modification in igration . Recombiepitopes  and woulP. falciparum, thus far, have only resulted in partial and temporary protection against malaria. Results of previous studies  have not revealed a protective antibody response against a single antigen of P. falciparum blood stages associated with clinical immunity in infected people but are compatible with responses to multiple blood-stage antigens by cytophilic antibodies (IgG1 and IgG3), with high affinity being more protective. Methods to assess antibody responses to multiple blood-stage antigens in previous studies included an analysis of responses to antigens generated by a microarray of recombinant antigens and a very limited analysis of responses to antigens separated by 1D or 2D electrophoresis by western blotting. Recombinant antigens lack posttranscriptional modification, which is found in more than 30% of immunorelevant antigens and do not contain isoforms of the same protein. It is therefore essential to develop an unbiased platform of 2D western blotting combined with the detection of antigens eliciting antibody responses with protective qualities (IgG1 and 3 subclasses and high affinity).Vaccines developed against P. falciparum are associated with clinical immunity to P. falciparum malaria. IgG2 or IgG4 responses are not associated with clinical immunity to P. falciparum malaria.IgG1 and IgG3 antibodies with high affinity to multiple schizont antigens of Objectives of this investigation were:P. falciparum schizont antigens, eliciting a protective immune response.To develop a method for comprehensive detection of all P. falciparum schizont antigens.To develop a method for the comprehensive detection of immunoglobulin subclass responses to To develop a method for the simultaneous quantification of the affinity of multiple antibodies to multiple antigens.P. falciparum malaria.To answer the question, whether high-affinity IgG1 and IgG3 antibodies to schizont antigens are associated with clinical immunity to P. falciparum parasitemia will be assessed for antiplasmodial immunoglobulin subclass responses with high affinity a total of 10 times during the establishment of the 2D western blot process. This requires taking 35 mL of blood from this patient. Alternatively, lyophilized pooled antiplasmodial hyperimmunoglobulin is reconstituted to give this amount with a dilution titrated to a physiological immunoglobulin concentration. By repeating the blotting process 10 times using the same antigen preparation and immunoglobulin donor (-pool) and comparing the results, an estimate of the intraindividual variability of western blot results will be enabled. Subsequently, a case-control study comparing antibody responses between clinically immune people and people suffering from severe P. falciparum malaria will be conducted. This study will provide a preliminary sample size calculation for larger seroepidemiological studies using immunorelevant parts of the immunome. The standard deviation of spot intensity on the image of the immunoblot and the corresponding quantity of antibody measured by immunochromatography for a protein with a known relationship to clinical immunity: IgG3 response and its affinity to merozoite surface protein-2 C-terminal antigen (MSP2-3D7) will be used for the ultimate sample size calculation, and the standard deviation for intraindividual intensity will have to be below the standard deviation used for sample size calculation for the assessment of interindividual differences between cases and controls.Owing to the lack of previous studies employing the methodology used in this investigation, there was no basis for a sample size calculation. The proposed project includes a pilot study that will inform future sample size calculations. To establish the methodology as a first step, the serum of a patient with afebrile P. falciparum hyperimmunoglobulin—is analyzed 10 times to be able to assess and achieve reproducibility, as mentioned earlier. After the successful establishment of the methodology, a group of 15 individuals with clinical immunity, defined as at least 10 years old, living in a holoendemic area for P. falciparum malaria from birth with at least four to five infections per year with asymptomatic P. falciparum malaria (parasitemia on antigen detection of histidine-rich protein or on microscopy) at the time of blood sampling is compared with 15 age-matched individuals from the same ethnic group and holoendemic area hospitalized with acute severe, complicated P. falciparum malaria .During the development of the methodology, the serum of a patient known to be clinically immune to malaria from a holoendemic area, that is, asymptomatic malaria using the most common definition—presence of parasites in peripheral thick blood smears, an axillary temperature <37.5°C, and an absence of malaria-related symptoms  or P. faP. falciparum parasitemia by microscopy or antigen detection is not documented, patients whose body temperature is not documented, patients with a known HIV infection, and patients with known acquired or congenital immunodeficiency are excluded.Patients in whom Ethical approval for the study will be sought from the National Research Ethics Service of the United Kingdom, and the study will be subject to institutional approval by the host institution. Informed written consent for the study will be sought from the participants of this study before its commencement.Immunorelevant antigens are identified by western blotting using participants’ serum after 2D electrophoresis of native schizont antigens with discrimination of protective antibody responses by the detection of antibody subclasses and added analysis of affinity. This is followed by the comparison of antibody responses of patients with severe malaria and clinically immune individuals. Protective antibody responses are defined as those of the IgG1 or IgG3 isotype and of greater magnitude or higher affinity in clinically immune individuals than in patients with severe malaria. Antigens against which such a response is detected are identified by using mass spectrometry .Plasmodium yoelii have been isolated and identified by immobilizing immunoglobulins from immune mice to a Sephadex column and then passing a blood-stage antigen mixture through the column followed by elution of antigens with sodium deoxycholate and identification of antigens after SDS-PAGE and western blotting with specific antibodies [Immunoaffinity chromatography involves the extraction of a specific antigen from an antigen mixture by using antibodies immobilized on a matrix . Immunoaffinity chromatography has been used to generate purified immunoglobulin subclass fractions from polyclonal human immunoglobulin preparations . This hatibodies ,47.Purified parasitophorous vacuolar membrane–enclosed merozoite structures (PEMSs) are initially generated, which contain a highly homogeneous synchronous parasite population at the mature schizont stage, which is essentially free of contaminating host cell proteins ,48,49. PP. falciparum parasitemia. This requires 25 2D-PAGE and western blot processes , including the generation of 25 2D-PAGE gels for protein identification and micropreparative purposes. For each patient or run of analysis, samples equivalent to 2700 μg of protein are required for nine 2D gels. Sufficient antigens is produced for all 25 runs of analysis. A total of at least 103.5 mg of P. falciparum protein is required for the project.The procedure is repeated until sufficient protein is generated for all gels required for the project. To ensure that the same protein mixture is used for all gels of this project, the isolated PEMSs are pooled after a sufficient amount is generated for all experiments. To facilitate equal conditions for processing of patient serum samples for cases and controls wherever possible, the serum of one patient with severe malaria is processed at the same time as the serum of an age-matched control with afebrile g for 60 min at 4°C [To determine how many PEMSs need to be produced, the protein content of the parasite pellet is determined by a test run of protein extraction. Pellets are pulse sonicated on a sonifier with microtip sonication  on ice for 10 min at 25% amplitude (with pulses of 2 seconds on and 3 seconds off), resulting in a 4-min total pulse-on time  (to prevn at 4°C . The supThe protocol regarding isoelectric focusing (IEF) that follows has essentially been adopted from a study by Goerg et al  Multime.Turn on the cooling system of Multiphor II  or immobilized pH gradient (IPG)phor .It is critical to prepare an equilibration buffer, which is used fresh each time; at room temperature, urea takes time to dissolve; therefore, start preparing the buffer before stopping the first-dimension run.The antigen mixture is subjected to 2D electrophoresis using IEF in the first dimension using a pH gradient of 3 to 11 in the IPG strip  and polyacrylamide gel electrophoresis using a large-format gel with a 26 cm×20 cm width and length in the second dimension .At this time point, the electrode paper strips (wicks) are replaced by fresh ones . VoltageAfter completion of IEF, IPG strips are equilibrated for 10 min each in the SDS equilibration buffer . The equilibrated IPG gel strips are slightly rinsed and blotted to remove the excess equilibration buffer. Finally, the strip is placed in the SDS electrophoresis running buffer  for 10 min as a final equilibration step . If not After IEF is completed, it is important to proceed immediately to gel equilibration, unless the IPG strip is being frozen for future analysis. Equilibration is always performed immediately before the second-dimension run, never before the storage of the Immobiline DryStrip gels. The second-dimension gel itself should be prepared and ready to accept the Immobiline DryStrip gel before beginning the equilibration protocol. SDS-PAGE in the second-dimension is performed using the Ettan DALTsix LargeVertical System . The preparations mentioned in this paragraph are performed before IEF is completed, followed by the preparation of electrophoresis .The total running time is approximately 9 hours. Nine gels are generated for each participant. Eight gels are used for immunoblotting and one preparative gel. After SDS-PAGE, the gel is withdrawn from the cassette and equilibrated in the transfer buffer: Towbin buffer consisting of 192 mM glycine, 25 mM Tris, 20% methanol (v/v), pH of 8.3 for 20 min to remove excess SDS. The membrane and the filter paper used for transfer are equilibrated in the transfer buffer for 10 min.Antigens from 8 gels are blotted onto the nitrocellulose membranes for each patient. Nitrocellulose is the medium of choice for these techniques because of its very high capacity for protein binding . The Ame2 [gentle treatment that does not interfere with the subsequent immunological detection steps  to avoid overheating during the blotting procedure . A sheet2 -55. The on steps .To allow binding of the antibodies in the serum to the blotted antigens, the bags are covered with glass plates on a shaker so that solutions are circulated properly within the packages by shaking overnight at 4°C . After i. Blot signals are normalized by relating to intensity of blot staining from a DyLight 800–conjugated antibody to a mouse monoclonal antibody of known concentration against the housekeeping antigen ovalbumin added to the PEMS antigens.For identification of high-avidity antibodies, the blotted antigen-antibody complexes on 4 of 8 nitrocellulose sheets per participant are subjected to a washing step with a dissociation buffer, including phosphate buffered saline (PBS) with 8 M urea with vigorous shaking for 5 min followed by 2 additional washes with the PBS buffer with vigorous shaking for 5 min. Avidity is quantified by the avidity index, which is the ratio of the optical density of urea-treated spots to the optical density value of untreated spots multiplied by 100, with all values higher than 50% ranked as high avidity. An avidity index of less than 30% is defined as low avidity and that of between 30% and 50% as intermediate avidity ,56. BounThe membrane is then exposed to Amersham ECL Select  and shaken for 1 min at room temperature. Excess fluid is dripped off the membranes . SubsequData generation from spots generated by the peroxidase reaction on the nitrocellulose membrane is performed using the ImageQuant TL  or MELANIE, PDQuest, Z3, Z4000, Phoretix, or Progenesis software package -61.master gel).One gel for each of the initial test runs and subsequently participants is subjected to a SYPRO Ruby staining for the localization of antigens identified as immunorelevant on a subsequent analysis of the 4 gels subjected to the analysis of isotype-specific high-affinity antibodies and used for a mass spectrometric analysis of gel sections with immunorelevant antigens (After the SYPRO Ruby staining (Molecular Probes), images could be generated using a Typhoon 9410 fluorescent imager set at an excitation bandwidth of 510 to 532 nm and an emission filter at 610 nm for matching.For mass spectrometry, gels selected for spot excision are incubated overnight in 350 mL of 10% (v/v) ethanol.m/z values are submitted to ProFound (Proteometrics software package) for peptide mass fingerprint searching, which queries a locally stored copy of the NCBInr database (version 20041201). Amino acid sequences found by this process can be attributed to proteins using the fully deciphered P. falciparum genome. In an alternative protocol, protein spots of interest are carefully excised from the SYPRO Ruby–stained gel, sliced into 1 mm2 pieces, and washed in 50% (v/v) ACN/25 mM ammonium bicarbonate, pH 7.8, and then dried in a vacuum concentrator. The digestion buffer  in 25 mM NH4HCO3 is added to the gel slices and incubated overnight at 37°C. The resulting peptides are extracted by the addition of 4 µL of water followed by 7 µL 30% (w/v) ACN/0.1% (v/v) TFA, vortexing, and brief centrifugation. The extracts are concentrated in a vacuum concentrator to approximately 5 µL. The concentrated sample extract is mixed in a ratio of 1:1 with a matrix  containing an internal standard  and 1 µL of the mixture is loaded onto a 96-position target. Peptide mass fingerprints are obtained semiautomatically on a MALDI mass spectrometer, and the resultant mass lists are searched against a nonredundant protein database (Swiss Prot/TrEMBL) using ProteinLynx V. 3.4 (Micromass) software. Protein identification was confirmed from a statistically significant MOWSE score resulting from fingerprint interrogation using MASCOT [P<.05) [Protein spots are cut from gels using a Genomic Solutions ProPic robot, and gel plugs are placed in 96-well plates in 100 mL of 10% ethanol. Proteins can be subjected to tryptic in-gel digestion on a ProGest workstation. To this end, the storage liquid was removed and the gel plugs were washed with ammonium bicarbonate  for 10 min and then with acetonitrile  for 10 min. Washing with ammonium bicarbonate and ACN is repeated. DTT (30 mL) is added to the gel plugs to reduce the proteins, and samples are incubated at 60°C for 10 min before they are allowed to cool to room temperature. The liquid is discarded and proteins are alkylated in iodoacetamide  for 45 min. Gel plugs are washed with ammonium bicarbonate and ACN twice before rehydration in 10 mL trypsin solution . After 10 min, ammonium bicarbonate  is added and samples are incubated at 37C for 4 hours. The reaction is stopped by the addition of 7 mL of 3% formic acid. A portion of the resulting supernatant is used for a matrix-assisted laser desorption/ionization/mass spectrometry (MALDI/MS) analysis. Samples can be spotted onto a MALDI target robotically (ProMS) using ZipTips. Peptides are eluted from the C18 (ZipTip) material with a matrix (acyano 4-hydroxy cinnamic acid) prepared in 60% ACN and 0.2% trifluoroacetic acid (TFA). MALDI/MS data are acquired on an Applied Biosystems Voyager DE-STR instrument, and the observed g MASCOT . Protein [P<.05) .The columns used for positive purification are modified by cross-linking of the antisubclass antibody to protein G using the Schneider cross-linking technique  reagent.Previously published data  suggesteFor each immunoglobulin subclass, 2 separate columns are generated (one for the antibody avidity assessment and one for the quantification of the antibody responses) using Protein G High Performance SpinTrap  per column suspended in 600 μL of lysis buffer  is loaded onto each pair of immunoglobulin subclass–loaded columns. Ovalbumin (50 µg) is added as a control antigen to all columns. Steps for binding are repeated (see section on purification), including the assessment of binding efficiency in the collection tubes. To assess the binding efficiency, antigen concentration in the collection tubes is measured by mass spectrometry.One each of the 2 immunoglobulin subclass columns is then subjected to an elution step with 1 M ammonium thiocyanate solution. The eluted antigen is quantified and identified by high pressure liquid chromatography/mass spectrometry (HPLC/MS).All columns are then subjected to elution with 1% deoxycholate to elute all bound antigens, which are then quantified and identified by HPLC/MS.P. falciparum infection. This is carried out by calculating the ratio to the level of antibodies to a mixture of all P. falciparum antigens and normalized by relating to the intensity of blot staining from a DyLight 800–conjugated antibody to a mouse monoclonal antibody of known concentration against the housekeeping antigen ovalbumin added to the PEMS antigens. If the corrected and normalized spot intensity for the antibody reaction to an antigen reveals a statistically more extensive antibody reaction of protective isotypes IgG1 or IgG3 (at least two-fold) and/or a reaction of higher affinity in clinically immune compared with nonimmune participants, this antigen is deemed to be immune-relevant and a candidate for future vaccine development. Principal component analysis (PCA) is used to visualize natural groupings in data based on similar patterns of variation. The presence of natural clusters in PCA indicates that there is more variation between groups than within groups. The absence of clusters indicates that the variation between groups is of the same order of magnitude as that within groups. This analysis will help identify patterns of antigens against which the immune reaction is protective.The western blot spot intensities are corrected for exposure of participants to On analysis of the results of the immunoaffinity chromatography, the relative antibody affinity is determined as the ratio of quantity of antigen eluted with and without a history of previous treatment with ammonium thiocyanate after normalization of the quantity relative to ovalbumin control. High affinity is hereby defined as the ratio being >50%, intermediate as 30% to 50%, and low as <30%. The antigens, together with their affinity bound by each IgG subclass, are then identified and registered.noise introduced by the aforementioned factors and image processing. This will increase the detection power of a statistically significant difference in antibody binding to specific antigens with a small sample size.The patterns emerging with this method are related to clinical immunity. A group of 15 comprising at least 10-year-old individuals with clinical immunity is compared with a group of 15 comprising at least 10-year-old age-matched patients from the same ethnic  group hospitalized with acute malaria. The association with a protective antibody response is derived from the quantitative data analysis of spot intensities. Given the small number of samples analyzed, it is essential to reduce the variability of antibody binding introduced by differences in antigen presence in the 2D gel, protein losses after transfer to the nitrocellulose membrane, differences in antibody binding due to washing steps, and differences in action/presence of chemicals present in different amounts locally denaturing the antibody binding sites on protein. To avoid the differences between cases and controls arising from these sources of variability, a pair of case and matching control is processed together under the same conditions, with the same batch of reagents and equipment under the same condition and at the same temperature. Data analysis is aimed at further reducing the effects of the There are two different types of errors in 2D gel image analysis data.First, there are spots that are not reproducibly observed (where detection or matching has failed) by the software. These are regarded as observation errors and account for a false discovery rate (FDR). Second, there are spots that are reproducibly observed, and their integrated optical density (IOD) has some intrinsic variations between the 2 gels. These are referred to as quantitation errors. To investigate the source and size of these errors, the errors between multiple scans are compared with the errors measured between multiple gels. The differences between these methods allow us to distinguish errors due to image analysis from errors particular to the gel running and staining system. This is done by comparing the variability of the detected protein load on the preparative gels in the 10 initial test runs as detected by SYPRO Ruby staining, with the variability of IOD on repeatedly running the densitometry scan. The other one is the comparison of western blot stain intensity between the initial run with the identical antibody source and the variability of results from scanning the same blot.Control for differences in detected quality and quantity of antiplasmodial antibody responses between groups due to different antigen uptake by gels and differences in handling of individual western blots affecting the binding of patient antibodies and secondary antibodies.Variability of signal due to different antigen uptake between gels, different binding of primary and secondary antibodies, and differences in staining related to methodology between the gels and not related to differences in the patient’s antibody responses are taken into account in the data analysis in the following ways.Variability due to processing between blots of cases and controls of the same run is corrected for by normalization by relating to the intensity of blot staining from a DyLight 800–conjugated antibody to a mouse monoclonal antibody of known concentration against the housekeeping antigen ovalbumin added to the PEMS antigens.Consensus western blot spot patterns are generated for clinically immune patients and patients with malaria. The consensus pattern is produced by spot detection on a fused image that essentially contains all spots in the experiments. Image fusion is a method that combines multiple images into a new, synthetic image, where each pixel is a function of the corresponding pixels in the input images. The resulting image looks like a real blot image, and more importantly, all spots from the experiment are represented on it. Thus, spots that are present only on a few of the gels can be located in the fused image and properly separated from those surrounding them. Software packages that can achieve this are used: PDQuest 2D analysis software (BIORAD) and Delta2D software version 4.0 (Decodon). A subgroup analysis is performed for different age groups. Antigens found to be associated with a protective antibody response can be identified by mass spectrometry using sections of the master gel where the antigen was found to be situated, from a comparison of the master gel with the position on the nitrocellulose membrane. To achieve this, the respective protein spots in the SYPRO Ruby–stained mastergel are identified after overlaying the western blot images used. PCA was used to identify potential outlier gels and outlier samples in the datasets. PCA is an excellent methodology for visualizing natural groupings or clusters in data based on similar patterns of variation. The presence of natural clusters in PCA, therefore, indicates that there is more variation between groups than within groups. The absence of clusters indicates that the variation between groups is of the same order of magnitude as that within groups. Two statistical criteria are used to select potential immunorelevant antigens: an FDR of 5% and a fold change of 2 or more. By limiting the FDR to 5%, it is ensured that minimal resources are spent on following up false-positive leads. A two-fold or greater change is chosen over a lower cutoff as it increases the chance that a change will be detectable with independent methods.P. falciparum antibodies. This is done by including a presentation of all the results as ratios to levels of global antiplasmodial antibodies. To address this, 50 µL of plasma from a patient is analyzed by Captia Malaria Total Antibody EIA (Trinity Biotech) at the Hospital of Tropical Diseases, London.One aim of this project is to consider that differences in the presence, quantity, and affinity of any specific antibody response between patients may simply be due to differences in intensity and duration of exposure and not due to the protective properties of such a response. To control for this fact, the antibody response needs to be related to the concentration of all P. falciparum was employed by Fontaine et al [P. falciparum mature stages (trophozoite and schizont stages) were enriched by Plasmion flotation. Immunoblots were directly digitalized using a Typhoon Trio Image scanner. Images were analyzed with Decyder v6.5 software, allowing accurate spot matching between 2D protein patterns and 2D antigenic patterns from gels and immunoblots, respectively. The sera allowed detection of 42 antigenic spots on the 2D immunoblot. The 42 antigenic spots detected on the immunoblot were excised and subjected to mass spectrometry for identification. The resulting fragment ion spectra were searched against the Homo sapiens and P. falciparum protein databases (NCBInr). Nineteen protein spots (45%) were successfully identified and corresponded to 13 distinct proteins according to their National Center for Biotechnology Information accession number. Among them, 4 were identified as P. falciparum proteins and 9 as Hsapiens proteins. This indicated the presence of autoantibodies in P. falciparum malaria. This study failed to identify several well-described P. falciparum antigenic proteins from the iRBC plasma membrane. The major part of these P. falciparum antigens are large hydrophobic proteins (>150 kDa), which are generally underrepresented and can be difficult to detect by 2D electrophoresis such as PfEMP1, Pf332 cytoadherence-linked asexual protein 9 (Clag 9) or EBA-175. The reducing and denaturing conditions used for the immunoblot analysis in this study did not consider conformational epitopes. Proteins with extreme isoelectric points or molecular weight may not have been detectable with the type of 2D electrophoresis applied in this study. 2D electrophoresis of P. falciparum schizonts performed with a high-resolution procedure revealed 661 protein spots in one study using a pH range of 4 to 7 in the first dimension. This indicated a loss of detection of proteins (there are 1036 predicted schizont proteins) at lower and higher pH which is relevant because of the bimodal distribution of isoelectric points of schizont proteins reaching those pH extremes [The first study applying 2D electrophoresis with western blotting to the serum of patients for an analysis of antibody responses to ne et al . The stuextremes .. This has been achieved by the use of monoclonal immunoglobulin subclass antibodies immobilized to Sephadex through proteins A and G. Antigens bound by immunoglobulins produced by mice with enhanced immunity to P yoelii have been isolated and identified by immobilizing immunoglobulin from immune mice to a Sephadex column and then passing a blood-stage antigen mixture through the column followed by elution of antigens with sodium deoxycholate and identification of antigens after SDS-PAGE and western blotting using specific antibodies.Immunoaffinity chromatography has been used to generate purified immunoglobulin subclass fractions from polyclonal human immunoglobulin preparations ,46. Thisprotein species [P. falciparum through glycosylation, phosphorylation, and methylation as well as the generation of disulfide bonds, which are all essential in the formation of nonlinear conformational epitopes not generated in recombinant antigens. The 2 methods employed for the identification of immunorelevant antigens have advantages and disadvantages and may thus complement each other.The approach to discovery of immunorelevant antigens proposed in this experimental protocol takes into account the complexity of native proteins with their numerous  species  for eachAdvantages of 2D western blotting of native antigens are as follows:protein species) generated by posttranscriptional modifications such as phosphorylation, glycosylation, and methylation.Potential for identification of antibody responses selective for specific defined isomeric forms of the same protein, including isoforms  antigens, which may lead to an underestimation of immune-relevant antigens that are not bound because of this process.The aforementioned methods may be suitable for the identification of hypothetical candidates for vaccine antigens. Only future prospective vaccine trials using the antigens identified could demonstrate that they elicit protective immunity. This proposed project is only geared to help identify candidates for such trials. A difficulty arising from the further use of native proteins or protein complexes as vaccine candidates is mass production. This has been brilliantly put in reference to merozoite protein-containing vaccines:In order to generate an immunogenic protein aggregate, it would be desirable to mimic the quaternary structure of merozoite proteins on the merozoite surface . Although there have been recent major advances in our knowledge of the protein-protein interactions of merozoite surface proteins, it is not yet possible to use this information to generate a homo- or heteropolymer to test as a vaccine candidate. Even with a more detailed picture of the ultrastructure of the merozoite surface coat, however, it would be technically difficult to produce a native merozoite surface protein complex for use in a vaccine consisting of recombinant proteins.


Despite vaccination recommendations, the burden of vaccine-preventable diseases remains high in older adults in the United States (US), contributing to substantial morbidity, mortality, and health care resource use and costs. To adequately plan for health care resource needs and to help inform vaccination policies, burden of disease projections that account for population aging over the coming decades are needed. As a first step, this exploratory study projects the burden of influenza, pertussis, herpes zoster, and pneumococcal disease in adults aged 50 y and older in the US, using a population-based modeling framework with separate decision trees for each vaccine-preventable disease. The model uses projected population estimates from the US Census Bureau to account for changes in the US population over time and then calculates expected numbers of cases and associated costs for each disease, keeping current estimates of age-specific disease incidence, vaccine coverage, and efficacy constant over time. This approach was used to focus the exploratory analysis on the burden of disease that may be expected due to population changes alone, assuming that all else remains unchanged. Due to population growth and the shifting age distribution over the next 30 y, the annual societal economic burden for the four vaccine-preventable diseases is projected to increase from approximately $35 billion to $49 billion, resulting in cumulative costs of approximately $1.3 trillion, as well as more than 1 million disease-related deaths. Given such notable burden, further efforts to increase vaccination coverage and effectiveness in older adults are needed. To adequately plan for future health care needs and to help inform vaccination policies, long-range projections of disease burden need to account for population growth and shifts in the US population age distribution . Although previous efforts have projected the burden of disease for HZ5 and pneumococcal disease6 separately, a consistent modeling framework has not yet been used to project the burden across diseases.Two previously published models have estimated the human and economic burden of these four vaccine-preventable diseases among US adults aged 50 y and older, with annual societal perspective cost estimates ranging from approximately 6.9 USD billion to 26.5 USD billion.The objective of this exploratory study was to use a consistent population-based modeling framework to project the clinical and economic burden of four vaccine-preventable diseases in US adults aged 50 y and older based on demographic changes alone .2 the underlying model structure consists of separate decision trees for each vaccine-preventable disease, accounting for disease severity and resource use. The decision tree model structures can be found in Supplementary Figures S1-S4.A population-based burden of disease model that accounts for the impact of population aging was built in Excel to explore the projected burden of influenza, pertussis, HZ, and pneumococcal disease in the US over a time horizon of up to 30 y. Consistent with the model by Ozawa et al.,7–10For each disease, the population is divided into an unvaccinated and a vaccinated population based on national estimates for vaccine coverage in 2016. Unvaccinated individuals are at risk for each disease, and vaccinated individuals are at reduced risk for each disease using corresponding vaccine efficacy and waning estimates . Case-severity distributions are included for each disease to appropriately capture costs of cases by severity. Individuals who develop cases of influenza, pertussis, and pneumococcal disease are at risk of disease-related death. Death from HZ is assumed negligible and is not modeled. The model assumes that vaccination reduces the probability of developing a case of disease, but does not affect the severity of disease if a case develops, despite evidence that vaccinated individuals may experience milder forms of disease as compared with unvaccinated individuals.11 to account for projected changes in the US population over 30 y (2017–2046). The model applies current estimates of disease incidence, disease-related mortality, vaccination coverage, vaccine efficacy, and disease-related costs  to isolate the impact of population aging on projected disease burden. Disease incidence was derived for unvaccinated individuals from surveillance data or taken from the published literature. Reported pertussis incidence from surveillance data was adjusted to account for underreporting by a factor of 100;12–16 this was tested in sensitivity analysis ranging the underreporting factor from 1 (no underreporting) to 200. Where possible, the model uses pneumococcal disease incidence data from 2010 to 2013  to isolate the impact of adult vaccination with PCV-13. A summary of disease-specific and vaccine-specific input parameters and ranges used in sensitivity analyses can be found in The model uses projected population estimates by 1-y age groups taken directly from the US Census Bureau1 focusing on the same four vaccine-preventable diseases among the same population of US adults aged 50 y and older; this population was also included in a subgroup analysis by Ozawa et al.2The modeled population is aligned with the model by McLaughlin et al.,In Year 1 (2017), the model starts with the US population of adults aged 50 y and older (stratified by 1-y age groups) and applies estimated disease incidence among unvaccinated individuals, as well as current estimated levels of vaccine coverage and efficacy to estimate the number of cases of each disease. In each subsequent year, projected estimates of the US population are used correspondingly to calculate numbers of cases of disease among individuals aged 50 y and older, holding all other inputs constant over time. By using these population projections, the model accounts for the aging of the population, as well as projected changes in all-cause mortality and net migration in each year. This approach also allows a new cohort of individuals who have just turned 50 y to “age into” the model in each year. 3Vaccines currently recommended by the ACIP for adults aged 50 y and older are presented in 40 Influenza vaccine protection is assumed to last one season and vaccine waning is not considered.For annual influenza vaccination, the model allows for a certain percentage of individuals aged 50 y or older to be vaccinated each year based on annual age-specific vaccine coverage rates from the National Immunization Survey and the Behavioral Risk Factor Surveillance System, using average coverage values over the past seven seasons (2010/2011 to 2016/2017).4 To account for these data and the assumption that vaccination coverage remains constant over time, the model uses an incremental approach to estimate the number of individuals vaccinated in a given year; the number of individuals “ever vaccinated” or “vaccinated in the last 10 years” in the modeled year are subtracted by the number of individuals estimated from the previous year, adjusting for deaths of vaccinated individuals. Each year, the model then calculates the proportion of vaccinated individuals with full or waned protection based on the number of individuals “ever vaccinated” or “vaccinated in the last 10 years” and the number of individuals vaccinated in the given year.For pertussis, HZ, and pneumococcal vaccines, the model estimates vaccine effectiveness using durations of protection and waning curves based on data from the published literature. These assumptions are summarized in Supplementary Table S6. For these vaccines, coverage data from the 2016 National Health Interview Survey are reported as the percentages of people who have ever received vaccination  or the percentage of people who have received vaccination in the last 10 y (for Tdap vaccine).42) with uniformly distributed uptake between 2006 and 2017. Beginning in Year 2 (2018), the model assumes that individuals aged 50 y and older received recombinant zoster vaccine , regardless of whether they had previously been vaccinated with ZVL. Of those vaccinated with RZV, 69% were assumed to receive the second dose.45 For pneumococcal vaccination, the model assumes that individuals aged 65 y and older began receiving both the conjugate and polysaccharide vaccines in 2014 (consistent with ACIP recommendations42) with uniformly distributed uptake between 2014 and present to calculate waning. Reported vaccine coverage for those who had “ever had a pneumonia shot” was tested in sensitivity analysis to test lower values for the percentage receiving both vaccines. For simplicity, the model excludes vaccination of high-risk individuals younger than 65 y of age.For pertussis vaccination, the model assumes that individuals aged 50 y and older who were previously vaccinated with Tdap in the last 10 y were uniformly distributed across the 10-y time frame, not taking into account changes in age recommendations over time . Our analysis focuses on the incremental protection provided by Tdap vaccine against pertussis compared with protection provided by tetanus diphtheria (Td) vaccine and does not include the burden of tetanus and diphtheria. As a simplifying assumption, the model also does not include the recommended booster doses every 10 y with either Td or Tdap vaccine. For HZ vaccination, the model assumes that individuals aged 60 y and older previously received the existing zoster vaccine live (ZVL) beginning in 2006  and undiscounted direct medical and societal perspective costs. Vaccination costs are not included. For the societal perspective, costs include direct medical costs incurred by payers and by patients , as well as disease-related indirect costs . The focus for this burden of disease analysis is on undiscounted results to appropriately capture the projected 46–48The human capital approach was used to estimate productivity loss costs due to disease-related mortality for each of the four modeled diseases, using mean annual age-specific market and nonmarket productivity estimates and age-specific estimates of remaining life expectancy.It is assumed that individuals can develop only one case of disease each year. Although some of the modeled diseases have long-term complications that may incur costs over multiple years , only costs in the first year of the disease are included.2 Additional details on model validation can be found in Supplementary Material S-3.The annual and cumulative disease burden is calculated overall and by age  over a time horizon of up to 30 y. In addition to base-case analyses, one-way sensitivity analyses were conducted for several model input parameters, varying each parameter up and down by a clinically plausible range  to assess the impact on total cost burden of disease. These clinically plausible ranges  were alsOver a 30-y period, the projected annual (undiscounted) number of cases of influenza, pertussis, HZ, and pneumococcal disease show trends of substantial growth , driven primarily by the 65+ y age category . Growth The cumulative burden of disease over the 30-y period is estimated to include approximately 270 million cases of influenza, 8 million cases of pertussis, 33 million cases of HZ, and 25 million cases of pneumococcal disease . The fouProjected burden of disease estimates and drivers of costs varied for each disease. For example, productivity loss costs due to disease morbidity and mortality ranged from approximately 8 USD billion for pertussis to almost 240 USD billion for pneumococcal disease . For infCosts of higher severity cases represented a significant portion of overall direct costs for all modeled diseases. For example, over 30 y, hospitalized influenza cases made up approximately 33% of all influenza cases, while associated direct costs of hospitalized cases (~200 USD billion) accounted for over 75% of all direct costs for treatment of influenza. Regarding HZ, over 20% of cases were considered complicated (with PHN or other non-pain complications) but made up over 58% of all direct medical costs for treating HZ. Inpatient pneumococcal cases represented only 37% of pneumococcal disease cases but contributed to over 85% of the direct costs attributed to the disease. Finally, this trend is also observed with pertussis, where the majority of direct costs were attributed to severe cases (over 80%) even though they only accounted for 7% of all cases (Supplementary Table S9).When clinically plausible ranges were explored for key input variables in one-way sensitivity analysis, the cumulative 30-y burden of disease from both the direct medical costs and societal perspective was most sensitive to changes in disease incidence . OverallWhen inputs for disease incidence, vaccination coverage, efficacy, and duration of protection were varied simultaneously using clinically plausible ranges, the projected burden of disease over a 30-y time horizon ranged from 388 USD billion to 1.1 USD trillion for the direct medical cost perspective and from 773 USD billion to 2.2 USD trillion for the societal perspective . MultiwaOver a 30-y period, the projected burden of influenza, pertussis, HZ, and pneumococcal disease in adults aged 50 y and older from the current study is substantial. The model predicted approximately 270 million cases of influenza, 8 million cases of pertussis, 33 million cases of HZ, and 25 million cases of pneumococcal disease, resulting in a cumulative total undiscounted cost of approximately 653 USD billion (425 USD billion discounted) from the direct cost perspective and 1.3 USD trillion (over 800 USD billion discounted) from the societal perspective. In the base-case analysis, the model assumed that disease incidence, disease-related mortality, vaccination coverage, and vaccine efficacy all remained constant; however, the projected annual number of cases of each disease grew over the 30-y period, driven by increases in population size due to the aging of the US population. Increases in the annual economic burden of disease from Year 1 to Year 30  outpaced the rate of overall growth in the population aged 50 y and older (36% increase). These results demonstrate a shift of the US population toward age groups that are more susceptible to disease and disease-related morbidity and mortality.Lower and upper bound estimates of disease burden from the one-way and multiway sensitivity analyses highlight the substantial burden of disease expected across the range of clinically plausible inputs for disease incidence, vaccine coverage, efficacy, and duration of protection. Results were most sensitive to model inputs related to disease incidence, as demonstrated by the one-way sensitivity analysis results. These results were driven primarily by the highly variable disease incidence across influenza seasons, with the lowest and highest incidence observed across seasons ranging nearly 10-fold. Although the projected burden of disease estimates have a high level of uncertainty over the 30-y time horizon , the projected cost of vaccine-preventable diseases is substantial even at the lower bound estimates.5 For influenza alone, our model predicts that cases will result in approximately 8 million hospitalizations, 4 million emergency department visits, and 107 million outpatient visits over the next several decades, which will need to be accommodated by health care facilities and providers. Efforts to reduce disease incidence and/or severity by policies aimed at increasing vaccination coverage and/or effectiveness of vaccines through research and funding priorities, as well as education of both medical providers and the general population on the importance and value of vaccination as a life-course approach, are needed to reduce the potential strain on the health care system and increase the population wellbeing and productivity over the next 30 years.The projected increase in vaccine-preventable disease burden among adults aged 50 y and older causes concern for the sustainability of existing health care provision over time.50 low vaccination rates among adults persist. Historically, vaccine coverage rates in the US have been lower among adults compared to children.51 This is likely due to a variety of factors that promote childhood vaccination, including state laws requiring vaccination for school entry and the federally funded Vaccines for Children Program.49 Lack of regularly scheduled well visits for adults, akin to “well child” visits that correspond with CDC recommendations for childhood vaccinations, may also be a differentiating factor; utilization of Medicare’s Annual Medicare Wellness Visit is associated with likelihood of vaccination.52 Patient out-of-pocket costs are another barrier to vaccination in older adults. Medicare beneficiaries may face significant co-pays or co-insurance for vaccines covered under Medicare Part D . Multiple studies have shown an association between increased patient out-of-pocket responsibility and reversal of vaccination claims (a proxy for no vaccination), both at the physician’s office and in the pharmacy setting.53–55Although the CDC and the Department of Health and Human Services provide information on strategies to increase adult vaccination rates ,56 a composite measure assessing compliance with all age-based recommended vaccines would be useful in monitoring overall adult vaccine program effectiveness.57 A composite vaccine measure including influenza, pneumococcal, Tdap, and HZ vaccines was successfully tested in clinics affiliated with the Indian Health Service,57 and an adult composite measure is currently being voluntarily reported in the 2020 HEDIS  measure set.58Implementation of additional quality measures for adult vaccination may be one solution to elevate the status of adult vaccination as an essential component of preventive care, much like childhood vaccination. While measures for adult influenza and pneumococcal vaccination exist in many quality measurement programs,59 The use of adjuvants, substances added to enhance immunogenicity, in vaccines for influenza60 and HZ33 have demonstrated potential for improving vaccine effectiveness in older adults. Additionally, research efforts to improve the durability of vaccine protection, such as those by the National Institute of Allergy and Infectious Diseases,61 the European Union,62 and private companies in pursuit of a “universal” influenza vaccine covering multiple influenza strains over multiple influenza seasons increase the potential to reduce disease burden, even at current vaccine coverage rates.In addition to improving vaccination coverage of currently available adult vaccines, further research and development efforts to improve vaccine effectiveness could also help reduce the projected burden of disease moving forward. Immunosenescence, the gradual deterioration of the immune system brought on by aging, results in older adults having less robust immune responses to vaccines.11 over the modeled time horizon. To meet the study objectives, the model analyses were conducted at the population level and did not involve additional complexities of tracking individual, single-year-of-age cohorts over time or modeling dynamic disease transmission. To estimate the effects of population aging on disease burden, the model also held other input parameter values constant over the modeled time horizon .The goal of this study was to use a consistent modeling framework across the four vaccine-preventable diseases to estimate the projected burden of disease over time at the population level. To maintain a simple model structure, population dynamics were implicitly captured by using cross-sectional projected population estimates from the US Census Bureau5 and pneumococcal disease6 over time but with several key differences as compared with our model. The model by Varghese et al.5 projected the potential burden of HZ in the US in the year 2030 by taking into consideration both trends in HZ incidence and population aging, but projections were based on HZ incidence rates before HZ vaccines became available. Wroe et al.6 projected the potential burden of pneumococcal pneumonia in the US between 2004 and 2040 based on projected population growth and shift in age distribution. However, this analysis only included pneumococcal pneumonia and did not consider all pneumococcal disease. Additionally, incidence data captured in the Wroe et al. model were from 2004, prior to the introduction of routine infant vaccination with PCV-13 that led to notable decreases in incidence among unvaccinated children and adults.To our knowledge, this is the first study to use a consistent modeling framework across diseases to project the burden of vaccine-preventable diseases among adults aged 50 y and older based solely on projected changes in the US population. Other studies have estimated the projected burden of HZThe model structure excluded some disease-specific details included in previous economic models  and did not account for pandemics or outbreaks. Although the input parameter values for pneumococcal disease implicitly captured costs and disease incidence for high-risk and low-risk populations aged 65 y and older, the input values and results were not modeled separately for these populations. The four vaccine-preventable diseases were also modeled over time separately and did not allow for interaction across the diseases .The model used aggregate-level vaccine coverage and average vaccine effectiveness values and waning functions that were not specific to vaccine products. For example, the effectiveness and cost of influenza vaccines represent an average across the available seasonal vaccines for adults. Similarly, for pertussis and pneumococcal vaccines, the effectiveness and costs are an average across products. The exception is HZ vaccines, where effectiveness and cost of ZVL are modeled in Year 1 (2017) and effectiveness and cost of RZV are modeled in subsequent years. These simplifying assumptions were made where the impact on the cost and clinical projections were deemed minor, while keeping the model structure and parameterization robust enough to address study objectives.63 While pneumococcal polysaccharide vaccine (PPSV-23) is still universally recommended, it is now recommended that the decision to give PCV-13 be made jointly between physician and patient. This change in PCV-13 recommendation was made in part due to the waning of PCV-13-serotype disease in the older adults after implementation of a universal pediatric recommendation, and from minimal population-level decreases in pneumonia and invasive pneumococcal disease since the adult recommendation in 2014. Results of our analysis may underestimate the burden of pneumococcal disease in adults aged 65 y and older if use of PCV-13 decreases substantially in the future. However, our analysis likely overestimates the burden of pneumococcal disease at younger ages because of the exclusion of pneumococcal vaccination in high-risk adults 50–64 y of age. Additionally, in October 2019, ACIP voted to revise the Td recommendation for decennial booster vaccination, allowing providers to administer either Td or Tdap every 10 y throughout life to ensure protection against tetanus and diphtheria and the option to also protect against pertussis.64 This change in Td recommendation was made to follow the widespread practice of administering Tdap instead of Td for booster vaccinations. Because the recommendation changed during the time frame of our model and booster doses were not modeled in the current analysis, our results may overestimate the burden of pertussis over the 30-y time horizon; however, the overestimation of the total burden of disease is likely to be relatively small given the small proportion (~1-2%) pertussis contributes to the overall burden.In 2019 ACIP made two relevant updates to the adult vaccination recommendations. In June 2019, ACIP voted to revise the pneumococcal vaccination recommendation for healthy individuals over 65 y of age.Maintaining current vaccine coverage rates among older adults is projected to lead to notable increases in influenza, pertussis, HZ, and pneumococcal disease burden over the next several decades due to population aging. Corresponding increases in the economic burden and health care resource utilization required to care for these vaccine-preventable diseases may cause increasing strain on the US health care system.Click here for additional data file.