Enhanced Immunogenicity and Protective Effects against SARS-CoV-2 Following Immunization with a Recombinant RBD-IgG Chimeric Protein.

The unprecedented global impact caused by SARS-CoV-2 imposed huge health and economic challenges, highlighting the urgent need for safe and effective vaccines. The receptor-binding domain (RBD) of SARS-CoV-2 is the major target for neutralizing antibodies and for vaccine formulations. Nonetheless, the low immunogenicity of the RBD requires the use of alternative strategies to enhance its immunological properties. Here, we evaluated the use of a subunit vaccine antigen generated after the genetic fusing of the RBD with a mouse IgG antibody. Subcutaneous administration of RBD-IgG led to the extended presence of the protein in the blood of immunized animals and enhanced RBD-specific IgG titers. Furthermore, RBD-IgG immunized mice elicited increased virus neutralizing antibody titers, measured both with pseudoviruses and with live original (Wuhan) SARS-CoV-2. Immunized K18-hACE2 mice were fully resistant to the lethal challenge of the Wuhan SARS-CoV-2, demonstrated by the control of body-weight loss and virus loads in their lungs and brains. Thus, we conclude that the genetic fusion of the RBD with an IgG molecule enhanced the immunogenicity of the antigen and the generation of virus-neutralizing antibodies, supporting the use of IgG chimeric antigens as an approach to improve the performance of SARS-CoV-2 subunit vaccines.

Diagnostic and vaccine potential of Zika virus envelope protein (E) derivates produced in bacterial and insect cells.

Introduction: In the present study we evaluated the features of different recombinant forms of Zika virus (ZIKV) proteins produced in either bacterial (Eschericha coli) or insect cells (Drosophila melanogaster). The ZIKV-envelope glycoprotein (EZIKV) is responsible for virus entry into host cells, is the main target of neutralizing antibodies and has been used as a target antigen either for serological tests or for the development of subunit vaccines. The EZIKV is composed of three structural and functional domains (EDI, EDII, and EDIII), which share extensive sequence conservation with the corresponding counterparts expressed by other flaviviruses, particularly the different dengue virus (DENV) subtypes. Methods: In this study, we carried out a systematic comparison of the antigenicity and immunogenicity of recombinant EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells. For the antigenicity analysis we collected 88 serum samples from ZIKV-infected participants and 57 serum samples from DENV-infected. For immunogenicity, C57BL/6 mice were immunized with two doses of EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells to evaluate humoral and cellular immune response. In addition, AG129 mice were immunized with EZIKV and then challenge with ZIKV. Results: Testing of samples collected from ZIKV-infected and DENV-infected participants demonstrated that the EZIKV and EDIIIZIKV produced in BL21 cells presented better sensitivity and specificity compared to proteins produced in S2 cells. In vivo analyses were carried out with C57BL/6 mice and the results indicated that, despite similar immunogenicity, antigens produced in S2 cells, particularly EZIKV and EDIIIZIKV, induced higher ZIKV-neutralizing antibody levels in vaccinated mice. In addition, immunization with EZIKV expressed in S2 cells delayed the onset of symptoms and increased survival rates in immunocompromised mice. All recombinant antigens, either produced in bacteria or insect cells, induced antigen-specific CD4+ and CD8+ T cell responses. Conclusion: In conclusion, the present study highlights the differences in antigenicity and immunogenicity of recombinant ZIKV antigens produced in two heterologous protein expression systems.

Antibody cross-reactivity and evidence of susceptibility to emerging Flaviviruses in the dengue-endemic Brazilian Amazon.

OBJECTIVES: Several Flaviviruses can co-circulate. Pre-existing immunity to one virus can modulate the response to a heterologous virus; however, the serological cross-reaction between these emerging viruses in dengue virus (DENV)-endemic regions are poorly understood. METHODS: A cross-sectional study was performed among the residents of Manaus city in the state of Amazonas, Brazil. The serological response was assessed by hemagglutination inhibition assay (HIA), enzyme-linked immunosorbent assay, and neutralization assay. RESULTS: A total of 74.52% of the participants were immunoglobulin G-positive (310/416), as estimated by lateral flow tests. Overall, 93.7% of the participants were seropositive (419/447) for at least one DENV serotype, and the DENV seropositivity ranged between 84.8% and 91.0%, as determined by HIA. About 93% had antiyellow fever virus 17D-reactive antibodies, whereas 80.5% reacted to wild-type yellow fever virus. Zika virus (ZIKV) had the lowest seropositivity percentage (52.6%) compared with other Flaviviruses. Individuals who were DENV-positive with high antibody titers by HIA or envelope protein domain III enzyme-linked immunosorbent assay reacted strongly with ZIKV, whereas individuals with low anti-DENV antibody titers reacted poorly toward ZIKV. Live virus neutralization assay with ZIKV confirmed that dengue serogroup and ZIKV-spondweni serogroup are far apart; hence, individuals who are DENV-positive do not cross-neutralize ZIKV efficiently. CONCLUSION: Taken together, we observed a high prevalence of DENV in the Manaus-Amazon region and a varying degree of cross-reactivity against emerging and endemic Flaviviruses. Epidemiological and exposure conditions in Manaus make its population susceptible to emerging and endemic arboviruses.

Reactivity of DENV-positive sera against recombinant envelope proteins produced in bacteria and eukaryotic cells.

Dengue is a mosquito-borne disease endemic in many tropical and subtropical countries. It is caused by the dengue virus (DENV) that can be classified into 4 different serotypes (DENV-1-4). Early diagnosis and management can reduce morbidity and mortality rates of severe forms of the disease, as well as decrease the risk of larger outbreaks. Hiperendemicity in some regions of the world and the possibility that some people develop a more severe form of disease after a secondary infection caused by antibody-dependent enhancement justify the need to understand more thoroughly the antibody response induced against the virus. Here, we successfully produced a recombinant DENV-2 envelope (E) protein and its domains (EDI/II and EDIII) in two distinct expression systems: the Drosophila S2 insect cell system and the BL21 (DE3) pLySs bacterial system. We then evaluated the reactivity of sera from patients previously infected with DENV to each recombinant protein and to each domain separately. Our results show that the E protein produced in Drosophila S2 cells is recognized more frequently than the protein produced in bacteria. However, the recognition of E protein produced in bacteria correlates better with the DENV-2 sera neutralization capacity. The results described here emphasize the differences observed when antigens produced in bacteria or eukaryotic cells are used and may be useful to gain more insight into the humoral immune responses induced by dengue infection.

STAT3 signaling modulates the immune response induced after antigen targeting to conventional type 1 dendritic cells through the DEC205 receptor.

Conventional dendritic cells (cDC) are a group of antigen-presenting cells specialized in priming T cell responses. In mice, splenic cDC are divided into conventional type 1 DC (cDC1) and conventional type 2 (cDC2). cDC1 are specialized to prime the Th1 CD4+ T cell response, while cDC2 are mainly associated with the induction of follicular helper T cell responses to support germinal center formation. However, the mechanisms that control the functions of cDC1 and cDC2 are not fully understood, especially the signaling pathways that can modulate their ability to promote different CD4+ T cell responses. Here, we targeted a model antigen for cDC1 and cDC2, through DEC205 and DCIR2 receptors, respectively, to study the role of the STAT3 signaling pathway in the ability of these cells to prime CD4+ T cells. Our results show that, in the absence of the STAT3 signaling pathway, antigen targeting to cDC2 induced similar frequencies of Tfh cells between STAT3-deficient mice compared to fully competent mice. On the other hand, Th1 and Th1-like Tfh cell responses were significantly reduced in STAT3-deficient mice after antigen targeting to cDC1 via the DEC205 receptor. In summary, our results indicate that STAT3 signaling does not control the ability of cDC2 to promote Tfh cell responses after antigen targeting via the DCIR2 receptor, but modulates the function of cDC1 to promote Th1 and Th1-like Tfh T cell responses after antigen targeting via the DEC205 receptor.

Validation of Serological Methods for COVID-19 and Retrospective Screening of Health Employees and Visitors to the São Paulo University Hospital, Brazil.

Reliable serological tests for the detection of SARS-CoV-2 antibodies among infected or vaccinated individuals are important for epidemiological and clinical studies. Low-cost approaches easily adaptable to high throughput screenings, such as Enzyme-Linked Immunosorbent Assays (ELISA) or electrochemiluminescence immunoassay (ECLIA), can be readily validated using different SARS-CoV-2 antigens. A total of 1,119 serum samples collected between March and July of 2020 from health employees and visitors to the University Hospital at the University of São Paulo were screened with the Elecsys® Anti-SARS-CoV-2 immunoassay (Elecsys) (Roche Diagnostics) and three in-house ELISAs that are based on different antigens: the Nucleoprotein (N-ELISA), the Receptor Binding Domain (RBD-ELISA), and a portion of the S1 protein (ΔS1-ELISA). Virus neutralization test (CPE-VNT) was used as the gold standard to validate the serological assays. We observed high sensitivity and specificity values with the Elecsys (96.92% and 98.78%, respectively) and N-ELISA (93.94% and 94.40%, respectively), compared with RBD-ELISA (90.91% sensitivity and 88.80% specificity) and the ΔS1-ELISA (77.27% sensitivity and 76% specificity). The Elecsys® proved to be a reliable SARS-CoV-2 serological test. Similarly, the recombinant SARS-CoV-2 N protein displayed good performance in the ELISA tests. The availability of reliable diagnostic tests is critical for the precise determination of infection rates, particularly in countries with high SARS-CoV-2 infection rates, such as Brazil. Collectively, our results indicate that the development and validation of new serological tests based on recombinant proteins may provide new alternatives for the SARS-CoV-2 diagnostic market.

STAT6 signaling pathway controls germinal center responses promoted after antigen targeting to conventional type 2 dendritic cells.

Conventional dendritic cells (cDCs) are antigen-presenting cells specialized in naïve T cell priming. Mice splenic cDCs are classified as cDC1s and cDC2s, and their main functions have been elucidated in the last decade. While cDC1s are specialized in priming type 1 helper T cells (TH1) and in cross presentation, cDC2s prime T follicular helper (TFH) cells that stimulate germinal center (GC) formation, plasma cell differentiation and antibody production. However, less is known about the molecular mechanisms used by cDCs to prime those responses. Here, using WT and STAT6-deficient mice (STAT6 KO), we targeted a model antigen to cDC1s and cDC2s via DEC205 and DCIR2 receptors, respectively, in an attempt to study whether the STAT6 signaling pathway would modulate cDCs' ability to prime helper T cells. We show that the differentiation and maturation of cDCs, after stimulation with an adjuvant, were comparable between WT and STAT6 KO mice. Besides, our results indicate that, in STAT6 KO mice, antigen targeting to cDC2s induced reduced TFH and GC responses, but did not alter plasma cells numbers and antibody titers. Thus, we conclude that the STAT6 signaling pathway modulates the immune response after antigen targeting to cDC2s via the DCIR2 receptor: while STAT6 stimulates the development of TFH cells and GC formation, plasma cell differentiation occurs in a STAT6 independent manner.

Intradermal Delivery of Dendritic Cell-Targeting Chimeric mAbs Genetically Fused to Type 2 Dengue Virus Nonstructural Protein 1.

Targeting dendritic cells (DCs) by means of monoclonal antibodies (mAbs) capable of binding their surface receptors (DEC205 and DCIR2) has previously been shown to enhance the immunogenicity of genetically fused antigens. This approach has been repeatedly demonstrated to enhance the induced immune responses to passenger antigens and thus represents a promising therapeutic and/or prophylactic strategy against different infectious diseases. Additionally, under experimental conditions, chimeric αDEC205 or αDCIR2 mAbs are usually administered via an intraperitoneal (i.p.) route, which is not reproducible in clinical settings. In this study, we characterized the delivery of chimeric αDEC205 or αDCIR2 mAbs via an intradermal (i.d.) route, compared the elicited humoral immune responses, and evaluated the safety of this potential immunization strategy under preclinical conditions. As a model antigen, we used type 2 dengue virus (DENV2) nonstructural protein 1 (NS1). The results show that the administration of chimeric DC-targeting mAbs via the i.d. route induced humoral immune responses to the passenger antigen equivalent or superior to those elicited by i.p. immunization with no toxic effects to the animals. Collectively, these results clearly indicate that i.d. administration of DC-targeting chimeric mAbs presents promising approaches for the development of subunit vaccines, particularly against DENV and other flaviviruses.

Conventional type 1 dendritic cells induce TH 1, TH 1-like follicular helper T cells and regulatory T cells after antigen boost via DEC205 receptor.

Conventional dendritic cells (cDCs) are specialized in antigen presentation. In the mouse spleen, cDCs are classified in cDC1s and cDC2s, and express DEC205 and DCIR2 endocytic receptors, respectively. Monoclonal antibodies (mAbs) αDEC205 (αDEC) and αDCIR2 have been fused to different antigens to deliver them to cDC1s or cDC2s. We immunized mice with αDEC and αDCIR2 fused to an antigen using Poly(I:C) as adjuvant. The initial immune response was analyzed from days 3 to 6 after the immunization. We also studied the influence of a booster dose. Our results showed that antigen targeting to cDC1s promoted a pro-inflammatory TH 1 cell response. Antigen targeting to cDC2s induced TFH cells, GCs, and plasma cell differentiation. After boost, antigen targeting to cDC1s improved the TH 1 cell response and induced TH 1-like TFH cells that led to an increase in specific antibody titers and IgG class switch. Additionally, a population of regulatory T cells was also observed. Antigen targeting to cDC2s did not improve the specific antibody response after boost. Our results add new information on the immune response induced after the administration of a booster dose with αDEC and αDCIR2 fusion mAbs. These results may be useful for vaccine design using recombinant mAbs.

Poly(I:C) Potentiates T Cell Immunity to a Dendritic Cell Targeted HIV-Multiepitope Vaccine.

Cellular immune responses are implicated in resistance to HIV and have been considered for the development of an effective vaccine. Despite their safety profile, subunit vaccines need to be delivered combined with an adjuvant. In the last years, in vivo antigen targeting to dendritic cells (DCs) using chimeric monoclonal antibodies (mAb) against the DC endocytic receptor DEC205/CD205 was shown to support long-term T cell immunity. Here, we evaluated the ability of different adjuvants to modulate specific cellular immune response when eight CD4+ HIV-derived epitopes (HIVBr8) were targeted to DEC205+ DCs in vivo. Immunization with two doses of αDECHIVBr8 mAb along with poly(I:C) induced Th1 cytokine production and higher frequency of HIV-specific polyfunctional and long-lived T cells than MPL or CpG ODN-assisted immunization. Although each adjuvant elicited responses against the 8 epitopes present in the vaccine, the magnitude of the T cell response was higher in the presence of poly(I:C). Moreover, poly(I:C) up regulated the expression of costimulatory molecules in both cDC1 and cDC2 DCs subsets. In summary, the use of poly(I:C) in a vaccine formulation that targets multiple epitopes to the DEC205 receptor improved the potency and the quality of HIV-specific responses when compared to other vaccine-adjuvant formulations. This study highlights the importance of the rational selection of antigen/adjuvant combination to potentiate the desired immune responses.

Dendritic Cell Targeting Using a DNA Vaccine Induces Specific Antibodies and CD4+ T Cells to the Dengue Virus Envelope Protein Domain III.

Dengue fever has become a global threat, causing millions of infections every year. An effective vaccine against all four serotypes of dengue virus (DENV) has not been developed yet. Among the different vaccination strategies available today, DNA vaccines are safe and practical, but currently induce relatively weak immune responses in humans. In order to improve immunogenicity, antigens may be targeted to dendritic cells (DCs), the main antigen presenting cells and orchestrators of the adaptive immune response, inducing T and B cell activation. It was previously shown that a DNA vaccine encoding a fusion protein comprised of an antigen and a single-chain Fv antibody (scFv) specific for the DC endocytic receptor DEC205 induced strong immune responses to the targeted antigen. In this work, we evaluate this strategy to improve the immunogenicity of dengue virus (DENV) proteins. Plasmids encoding the scFv αDEC205, or an isotype control (scFv ISO), fused to the DENV2 envelope protein domain III (EDIII) were generated, and EDIII specific immune responses were evaluated in immunized mice. BALB/c mice were intramuscularly (i.m.) immunized three times with plasmid DNAs encoding either scDEC-EDIII or scISO-EDIII followed by electroporation. Analyses of the antibody responses indicated that EDIII fusion with scFv targeting the DEC205 receptor significantly enhanced serum anti-EDIII IgG titers that inhibited DENV2 infection. Similarly, mice immunized with the scDEC-EDIII plasmid developed a robust CD4+ T cell response to the targeted antigen, allowing the identification of two linear epitopes recognized by the BALB/c haplotype. Taken together, these results indicate that targeting DENV2 EDIII protein to DCs using a DNA vaccine encoding the scFv αDEC205 improves both antibody and CD4+ T cell responses. This strategy opens perspectives for the use of DNA vaccines that encode antigens targeted to DCs as a strategy to increase immunogenicity.

Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin.

The circumsporozoite protein (CSP) is the major surface protein of malaria sporozoites (SPZs), the motile and invasive parasite stage inoculated in the host skin by infected mosquitoes. Antibodies against the central CSP repeats of different plasmodial species are known to block SPZ infectivity1-5, but the precise mechanism by which these effectors operate is not completely understood. Here, using a rodent Plasmodium yoelii malaria model, we show that sterile protection mediated by anti-P. yoelii CSP humoral immunity depends on the parasite inoculation into the host skin, where antibodies inhibit motility and kill P. yoelii SPZs via a characteristic 'dotty death' phenotype. Passive transfer of an anti-repeat monoclonal antibody (mAb) recapitulates the skin inoculation-dependent protection, in a complement- and Fc receptor γ-independent manner. This purified mAb also decreases motility and, notably, induces the dotty death of P. yoelii SPZs in vitro. Cytotoxicity is species-transcendent since cognate anti-CSP repeat mAbs also kill Plasmodium berghei and Plasmodium falciparum SPZs. mAb cytotoxicity requires the actomyosin motor-dependent translocation and stripping of the protective CSP surface coat, rendering the parasite membrane susceptible to the SPZ pore-forming-like protein secreted to wound and traverse the host cell membrane6. The loss of SPZ fitness caused by anti-P. yoelii CSP repeat antibodies is thus a dynamic process initiated in the host skin where SPZs either stop moving7, or migrate and traverse cells to progress through the host tissues7-9 at the eventual expense of their own life.

CpG Oligodeoxinucleotides and Flagellin Modulate the Immune Response to Antigens Targeted to CD8α+ and CD8α- Conventional Dendritic Cell Subsets.

Dendritic cells (DCs) are antigen-presenting cells essential for the induction of adaptive immune responses. Their unprecedented ability to present antigens to T cells has made them excellent targets for vaccine development. In the last years, a new technology based on antigen delivery directly to different DC subsets through the use of hybrid monoclonal antibodies (mAbs) to DC surface receptors fused to antigens of interest opened new perspectives for the induction of robust immune responses. Normally, the hybrid mAbs are administered with adjuvants that induce DC maturation. In this work, we targeted an antigen to the CD8α+ or the CD8α- DC subsets in the presence of CpG oligodeoxinucleotides (ODN) or bacterial flagellin, using hybrid αDEC205 or αDCIR2 mAbs, respectively. We also accessed the role of toll-like receptors (TLRs) 5 and 9 signaling in the induction of specific humoral and cellular immune responses. Wild-type and TLR5 or TLR9 knockout mice were immunized with two doses of the hybrid αDEC205 or αDCIR2 mAbs, as well as with an isotype control, together with CpG ODN 1826 or flagellin. A chimeric antigen containing the Plasmodium vivax 19 kDa portion of the merozoite surface protein (MSP119) linked to the Pan-allelic DR epitope was fused to each mAb. Specific CD4+ T cell proliferation, cytokine, and antibody production were analyzed. We found that CpG ODN 1826 or flagellin were able to induce CD4+ T cell proliferation, CD4+ T cells producing pro-inflammatory cytokines, and specific antibodies when the antigen was targeted to the CD8α+ DC subset. On the other hand, antigen targeting to CD8α- DC subset promoted specific antibody responses and proliferation, but no detectable pro-inflammatory CD4+ T cell responses. Also, specific antibody responses after antigen targeting to CD8α+ or CD8α- DCs were reduced in the absence of TLR9 or TLR5 signaling, while CD4+ T cell proliferation was mainly affected after antigen targeting to CD8α+ DCs and in the absence of TLR9 signaling. These results extend our understanding of the modulation of specific immune responses induced by antigen targeting to DCs in the presence of different adjuvants. Such knowledge may be useful for the optimization of DC-based vaccines.

Dendritic Cell Targeting Effectively Boosts T Cell Responses Elicited by an HIV Multiepitope DNA Vaccine.

Despite several efforts in the last decades, an efficacious HIV-1 vaccine is still not available. Different approaches have been evaluated, such as recombinant proteins, viral vectors, DNA vaccines, and, most recently, dendritic cell (DC) targeting. This strategy is based on DC features that place them as central for induction of immunity. Targeting is accomplished by the use of chimeric monoclonal antibodies directed to DC surface receptors fused to the antigen of interest. In this work, we targeted eight promiscuous HIV-derived CD4+ T cell epitopes (HIVBr8) to the DEC205+ DCs by fusing the multiepitope immunogen to the heavy chain of αDEC205 (αDECHIVBr8), in the presence of the TLR3 agonist poly (I:C). In addition, we tested a DNA vaccine encoding the same epitopes using homologous or heterologous prime-boost regimens. Our results showed that mice immunized with αDECHIVBr8 presented higher CD4+ and CD8+ T cell responses when compared to mice that received the DNA vaccine (pVAXHIVBr8). In addition, pVAXHIVBr8 priming followed by αDECHIVBr8 boosting induced higher polyfunctional proliferative and cytokine-producing T cell responses to HIV-1 peptides than homologous DNA immunization or heterologous αDEC prime/DNA boost. Based on these results, we conclude that homologous prime-boost and heterologous boosting immunization strategies targeting CD4+ epitopes to DCs are effective to improve HIV-specific cellular immune responses when compared to standalone DNA immunization. Moreover, our results indicate that antigen targeting to DC is an efficient strategy to boost immunity against a multiepitope immunogen, especially in the context of DNA vaccination.

HIV Envelope Trimer Specific Immune Response Is Influenced by Different Adjuvant Formulations and Heterologous Prime-Boost.

The development of a preventive vaccine against human immunodeficiency virus (HIV-1) infection is the most efficient method to control the epidemic. The ultimate goal is to develop a vaccine able to induce specific neutralizing, non-neutralizing antibodies and cellular mediated immunity (CMI). Humoral and CMI responses can be directed to glycoproteins that are normally presented as a trimeric spike on the virus surface (gp140). Despite safer, subunit vaccines are normally less immunogenic/effective and need to be delivered together with an adjuvant. The choice of a suitable adjuvant can induce effective humoral and CMI that utterly lead to full protection against disease. In this report, we established a hierarchy of adjuvant potency on humoral and CMI when admixed with the recombinant HIV gp140 trimer. We show that vaccination with gp140 in the presence of different adjuvants can induce high-affinity antibodies, follicular helper T cells and germinal center B cells. The data show that poly (I:C) is the most potent adjuvant to induce specific CMI responses evidenced by IFN-γ production and CD4+/CD8+ T cell proliferation. Furthermore, we demonstrate that combining some adjuvants like MPL plus Alum and MPL plus MDP exert additive effects that impact on the magnitude and quality of humoral responses while mixing MDP with poly (I:C) or with R848 had no impact on total IgG titers but highly impact IgG subclass. In addition, heterologous DNA prime- protein boost yielded higher IgG titers when compare to DNA alone and improved the quality of humoral response when compare to protein immunization as evidenced by IgG1/IgG2a ratio. The results presented in this paper highlight the importance of selecting the correct adjuvant-antigen combination to potentiate desired cells for optimal stimulation.

Variant proteins of Plasmodium vivax are not clonally expressed in natural infections.

Plasmodium vivax is the most widely distributed human malaria parasite and responsible for 70-80 million clinical cases each year and a large socio-economical burden. The sequence of a chromosome end from P. vivax revealed the existence of a multigene superfamily, termed vir (P. vivax variant antigens), that can be subdivided into different subfamilies based on sequence similarity analysis and which represents close to 10-20% of the coding sequences of the parasite. Here we show that there is a vast repertoire of vir genes abundantly expressed in isolates obtained from human patients, that different vir gene subfamilies are transcribed in mature asexual blood stages by individual parasites, that VIR proteins are not clonally expressed and that there is no significant difference in the recognition of VIR-tags by immune sera of first-infected patients compared with sera of multiple-infected patients. These data provide to our knowledge the first comprehensive study of vir genes and their encoding variant proteins in natural infections and thus constitute a baseline for future studies of this multigene superfamily. Moreover, whereas our data are consistent with a major role of vir genes in natural infections, they are inconsistent with a predominant role in the strict sense of antigenic variation.