Age-Related Changes in the Nasopharyngeal Microbiome Are Associated With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection and Symptoms Among Children, Adolescents, and Young Adults.

BACKGROUND: Children are less susceptible to SARS-CoV-2 infection and typically have milder illness courses than adults, but the factors underlying these age-associated differences are not well understood. The upper respiratory microbiome undergoes substantial shifts during childhood and is increasingly recognized to influence host defense against respiratory pathogens. Thus, we sought to identify upper respiratory microbiome features associated with SARS-CoV-2 infection susceptibility and illness severity. METHODS: We collected clinical data and nasopharyngeal swabs from 285 children, adolescents, and young adults (<21 years) with documented SARS-CoV-2 exposure. We used 16S ribosomal RNA gene sequencing to characterize the nasopharyngeal microbiome and evaluated for age-adjusted associations between microbiome characteristics and SARS-CoV-2 infection status and respiratory symptoms. RESULTS: Nasopharyngeal microbiome composition varied with age (PERMANOVA, P < .001; R2 = 0.06) and between SARS-CoV-2-infected individuals with and without respiratory symptoms (PERMANOVA, P  = .002; R2 = 0.009). SARS-CoV-2-infected participants with Corynebacterium/Dolosigranulum-dominant microbiome profiles were less likely to have respiratory symptoms than infected participants with other nasopharyngeal microbiome profiles (OR: .38; 95% CI: .18-.81). Using generalized joint attributed modeling, we identified 9 bacterial taxa associated with SARS-CoV-2 infection and 6 taxa differentially abundant among SARS-CoV-2-infected participants with respiratory symptoms; the magnitude of these associations was strongly influenced by age. CONCLUSIONS: We identified interactive relationships between age and specific nasopharyngeal microbiome features that are associated with SARS-CoV-2 infection susceptibility and symptoms in children, adolescents, and young adults. Our data suggest that the upper respiratory microbiome may be a mechanism by which age influences SARS-CoV-2 susceptibility and illness severity.

Age-related changes in the upper respiratory microbiome are associated with SARS-CoV-2 susceptibility and illness severity.

Children are less susceptible to SARS-CoV-2 and typically have milder illness courses than adults. We studied the nasopharyngeal microbiomes of 274 children, adolescents, and young adults with SARS-CoV-2 exposure using 16S rRNA gene sequencing. We find that higher abundances of Corynebacterium species are associated with SARS-CoV-2 infection and SARS-CoV-2-associated respiratory symptoms, while higher abundances of Dolosigranulum pigrum are present in SARS-CoV-2-infected individuals without respiratory symptoms. We also demonstrate that the abundances of these bacteria are strongly, and independently, associated with age, suggesting that the nasopharyngeal microbiome may be a potentially modifiable mechanism by which age influences SARS-CoV-2 susceptibility and severity. SUMMARY: Evaluation of nasopharyngeal microbiome profiles in children, adolescents, and young adults with a SARS-CoV-2-infected close contact identified specific bacterial species that vary in abundance with age and are associated with SARS-CoV-2 susceptibility and the presence of SARS-CoV-2-associated respiratory symptoms.

Severe Acute Respiratory Syndrome Coronavirus 2 Infections Among Children in the Biospecimens from Respiratory Virus-Exposed Kids (BRAVE Kids) Study.

BACKGROUND: Child with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection typically have mild symptoms that do not require medical attention, leaving a gap in our understanding of the spectrum of SARS-CoV-2-related illnesses that the viruses causes in children. METHODS: We conducted a prospective cohort study of children and adolescents (aged <21 years) with a SARS-CoV-2-infected close contact. We collected nasopharyngeal or nasal swabs at enrollment and tested for SARS-CoV-2 using a real-time polymerase chain reaction assay. RESULTS: Of 382 children, 293 (77%) were SARS-CoV-2-infected. SARS-CoV-2-infected children were more likely to be Hispanic (P < .0001), less likely to have asthma (P = .005), and more likely to have an infected sibling contact (P = .001) than uninfected children. Children aged 6-13 years were frequently asymptomatic (39%) and had respiratory symptoms less often than younger children (29% vs 48%; P = .01) or adolescents (29% vs 60%; P < .001). Compared with children aged 6-13 years, adolescents more frequently reported influenza-like (61% vs 39%; P < .001) , and gastrointestinal (27% vs 9%; P = .002), and sensory symptoms (42% vs 9%; P < .0001) and had more prolonged illnesses (median [interquartile range] duration: 7 [4-12] vs 4 [3-8] days; P = 0.01). Despite the age-related variability in symptoms, wWe found no difference in nasopharyngeal viral load by age or between symptomatic and asymptomatic children. CONCLUSIONS: Hispanic ethnicity and an infected sibling close contact are associated with increased SARS-CoV-2 infection risk among children, while asthma is associated with decreased risk. Age-related differences in clinical manifestations of SARS-CoV-2 infection must be considered when evaluating children for coronavirus disease 2019 and in developing screening strategies for schools and childcare settings.

SARS-CoV-2 Infections Among Children in the Biospecimens from Respiratory Virus-Exposed Kids (BRAVE Kids) Study.

BACKGROUND: Children with SARS-CoV-2 infection typically have mild symptoms that do not require medical attention, leaving a gap in our understanding of the spectrum of illnesses that the virus causes in children. METHODS: We conducted a prospective cohort study of children and adolescents (<21 years of age) with a SARS-CoV-2-infected close contact. We collected nasopharyngeal or nasal swabs at enrollment and tested for SARS-CoV-2 using a real-time PCR assay. RESULTS: Of 382 children, 289 (76%) were SARS-CoV-2-infected. SARS-CoV-2-infected children were more likely to be Hispanic (p<0.0001), less likely to have a history of asthma (p=0.009), and more likely to have an infected sibling contact (p=0.0007) than uninfected children. Children ages 6-13 years were frequently asymptomatic (38%) and had respiratory symptoms less often than younger children (30% vs. 49%; p=0.008) or adolescents (30% vs. 59%; p<0.0001). Compared to children ages 6-13 years, adolescents more frequently reported influenza-like (61% vs. 39%; p=0.002), gastrointestinal (26% vs. 9%; p=0.003), and sensory symptoms (43% vs. 9%; p<0.0001), and had more prolonged illnesses [median (IQR) duration: 7 (4, 12) vs. 4 (3, 8) days; p=0.004]. Despite the age-related variability in symptoms, we found no differences in nasopharyngeal viral load by age or between symptomatic and asymptomatic children. CONCLUSIONS: Hispanic ethnicity and an infected sibling close contact are associated with increased SARS-CoV-2 infection risk among children, while a history of asthma is associated with decreased risk. Age-related differences in the clinical manifestations of SARS-CoV-2 infection must be considered when evaluating children for COVID-19 and in developing screening strategies for schools and childcare settings.

HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces gp41 Antibody Immunodominance in Rhesus Macaques.

Dominant antibody responses in vaccinees who received the HIV-1 multiclade (A, B, and C) envelope (Env) DNA/recombinant adenovirus virus type 5 (rAd5) vaccine studied in HIV-1 Vaccine Trials Network (HVTN) efficacy trial 505 (HVTN 505) targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs), which are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells than gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy trial. These data demonstrated that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response.IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and neonatal RMs indicated that early vaccination could not overcome gp41 dominant responses.

Plasmablast Response to Primary Rhesus Cytomegalovirus (CMV) Infection in a Monkey Model of Congenital CMV Transmission.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and the leading infectious cause of neurologic deficits and hearing loss in newborns. Development of a maternal HCMV vaccine to prevent vertical virus transmission is a high priority, yet protective maternal immune responses following acute infection are poorly understood. To characterize the maternal humoral immune response to primary CMV infection, we investigated the plasmablast and early antibody repertoire using a nonhuman primate model with two acutely rhesus CMV (RhCMV)-infected animals-a CD4+ T cell-depleted dam that experienced fetal loss shortly after vertical RhCMV transmission and an immunocompetent dam that did not transmit RhCMV to her infant. Compared to the CD4+ T cell-depleted dam that experienced fetal loss, the immunocompetent, nontransmitting dam had a more rapid and robust plasmablast response that produced a high proportion of RhCMV-reactive antibodies, including the first identified monoclonal antibody specific for soluble and membrane-associated RhCMV envelope glycoprotein B (gB). Additionally, we noted that plasmablast RhCMV-specific antibodies had variable gene usage and maturation similar to those observed in a monkey chronically coinfected with simian immunodeficiency virus (SIV) and RhCMV. This study reveals characteristics of the early maternal RhCMV-specific humoral immune responses to primary RhCMV infection in rhesus monkeys and may contribute to a future understanding of what antibody responses should be targeted by a vaccine to eliminate congenital HCMV transmission. Furthermore, the identification of an RhCMV gB-specific monoclonal antibody underscores the possibility of modeling future HCMV vaccine strategies in this nonhuman primate model.

Tissue memory B cell repertoire analysis after ALVAC/AIDSVAX B/E gp120 immunization of rhesus macaques.

The ALVAC prime/ALVAC + AIDSVAX B/E boost RV144 vaccine trial induced an estimated 31% efficacy in a low-risk cohort where HIV­1 exposures were likely at mucosal surfaces. An immune correlates study demonstrated that antibodies targeting the V2 region and in a secondary analysis antibody-dependent cellular cytotoxicity (ADCC), in the presence of low envelope-specific (Env-specific) IgA, correlated with decreased risk of infection. Thus, understanding the B cell repertoires induced by this vaccine in systemic and mucosal compartments are key to understanding the potential protective mechanisms of this vaccine regimen. We immunized rhesus macaques with the ALVAC/AIDSVAX B/E gp120 vaccine regimen given in RV144, and then gave a boost 6 months later, after which the animals were necropsied. We isolated systemic and intestinal vaccine Env-specific memory B cells. Whereas Env-specific B cell clonal lineages were shared between spleen, draining inguinal, anterior pelvic, posterior pelvic, and periaortic lymph nodes, members of Env­specific B cell clonal lineages were absent in the terminal ileum. Env­specific antibodies were detectable in rectal fluids, suggesting that IgG antibodies present at mucosal sites were likely systemically produced and transported to intestinal mucosal sites.

Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection in vivo.

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

Rapid Development of gp120-Focused Neutralizing B Cell Responses during Acute Simian Immunodeficiency Virus Infection of African Green Monkeys.

UNLABELLED: The initial phases of acute human immunodeficiency virus type 1 (HIV-1) infection may be critical for development of effective envelope (Env)-specific antibodies capable of impeding the establishment of the latent pool of HIV-1-infected CD4(+) T cells, preventing virus-induced immune hyperactivation to limit disease progression and blocking vertical virus transmission. However, the initial systemic HIV-1 Env-specific antibody response targets gp41 epitopes and fails to control acute-phase viremia. African-origin, natural simian immunodeficiency virus (SIV) hosts do not typically progress to AIDS and rarely postnatally transmit virus to their infants, despite high milk viral loads. Conversely, SIV-infected rhesus macaques (RMs), Asian-origin nonnatural SIV hosts, sustain pathogenic SIV infections and exhibit higher rates of postnatal virus transmission. In this study, of acute SIV infection, we compared the initial systemic Env-specific B cell responses of AGMs and RMs in order to probe potential factors influencing the lack of disease progression observed in AGMs. AGMs developed higher-magnitude plasma gp120-specific IgA and IgG responses than RMs, whereas RMs developed more robust gp140-directed IgG responses. These gp120-focused antibody responses were accompanied by rapid autologous neutralizing responses during acute SIV infection in AGMs compared to RMs. Moreover, acute SIV infection elicited a higher number of circulating Env-specific memory B cells in peripheral blood of AGMs than in the blood of RMs. These findings indicate that AGMs have initial systemic Env-specific B cell responses to SIV infection distinct from those of a nonnatural SIV host, resulting in more functional SIV-specific humoral responses, which may be involved in impairing pathogenic disease progression and minimizing postnatal transmission. IMPORTANCE: Due to the worldwide prevalence of HIV-1 infections, development of a vaccine to prevent infection or limit the viral reservoir remains an important goal. HIV-1-infected humans, as well as SIV-infected nonnatural SIV hosts, develop pathogenic infections and readily transmit the virus to their infants. Conversely, natural SIV hosts do not develop pathogenic infections and rarely transmit the virus to their infants. The immunologic factors contributing to these favorable outcomes in natural SIV hosts could prove invaluable for directing HIV-1 vaccine and therapy design. This study identified distinctions in the specificity and function of the initial systemic SIV envelope-specific B cell response that developed during acute SIV infection in natural and nonnatural SIV host species. Identification of distinct acute B cell responses in natural SIV hosts may inform vaccine strategies seeking to elicit similar responses prior to or during the initial phases of acute HIV-1 infection.

Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.

Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1-transmitting mothers and 165 propensity score-matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1-infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3-specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.

Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements.

B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual's secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.

HIV-1 vaccine-induced C1 and V2 Env-specific antibodies synergize for increased antiviral activities.

The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission. Importance: The Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design.

Toll-like receptor 7/8 (TLR7/8) and TLR9 agonists cooperate to enhance HIV-1 envelope antibody responses in rhesus macaques.

UNLABELLED: The development of a vaccine that can induce high titers of functional antibodies against HIV-1 remains a high priority. We have developed an adjuvant based on an oil-in-water emulsion that incorporates Toll-like receptor (TLR) ligands to test whether triggering multiple pathogen-associated molecular pattern receptors could enhance immunogenicity. Compared to single TLR agonists or other pairwise combinations, TLR7/8 and TLR9 agonists combined were able to elicit the highest titers of binding, neutralizing, and antibody-dependent cellular cytotoxicity-mediating antibodies against the protein immunogen, transmitted/founder HIV-1 envelope gp140 (B.63521). We further found that the combination of TLR7/8 and TLR9 agonists was associated with the release of CXCL10 (IP-10), suggesting that this adjuvant formulation may have optimally stimulated innate and adaptive immunity to elicit high titers of antibodies. IMPORTANCE: Combining TLR agonists in an adjuvant formulation resulted in higher antibody levels compared to an adjuvant without TLR agonists. Adjuvants that combine TLR agonists may be useful for enhancing antibody responses to HIV-1 vaccines.

Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene family.

The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.

HIV-1 gp120 vaccine induces affinity maturation in both new and persistent antibody clonal lineages.

Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120(W6.1D)). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of V(H) somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120(W6.1D) was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination.

BACKGROUND: During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. METHODS AND FINDINGS: To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. CONCLUSION: The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

Polyclonal B cell differentiation and loss of gastrointestinal tract germinal centers in the earliest stages of HIV-1 infection.

BACKGROUND: The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+) T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells. METHODS AND FINDINGS: In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis. CONCLUSIONS: Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.

Regulation of maturation and activating potential in CD8+ versus CD8- dendritic cells following in vivo infection with vaccinia virus.

DC maturation is known to be a necessary step in the generation of an effective immune response. We have used vaccinia virus (VACV) as a model to investigate the regulation of DC subsets in vivo following infection. While a number of in vitro studies have shown that DC infected with VACV fail to undergo maturation, the effect of VACV infection on the maturation of and cytokine production by DC subsets in vivo remains less defined. We have found that following systemic infection with vaccinia virus, both CD8+ and CD8- dendritic cells are infected. The number of infected DC peaked at 6 h and was highly decreased by 24 h post-infection. In both subsets, there was evidence of generalized upregulation of costimulatory molecules. Surprisingly, this included vaccinia infected DC, suggesting the regulation of DC maturation in vivo is much more complex and likely influenced by DC extrinsic signals. However, while we observed generalized upregulation of costimulatory molecules, IL-12 production was restricted to a subset of non-infected cells in both the CD8+ and CD8- DC populations. Importantly, the control of IL-12 production was differentially dependent on MyD88 signaling. IL-12 production was ablated in the absence of MyD88 in CD8- DC, while it was unchanged in CD8+ DC. These findings provide new insights into the control of DC maturation in vivo and demonstrate that the regulation of maturation in vivo following virus infection can be differentially controlled in distinct types of DC.