Biology subjects

Permar, S. R.

Publications and source records attributed to Permar, S. R..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

A single, improbable B cell receptor mutation confers potent neutralization against cytomegalovirus

Cytomegalovirus (CMV) is a leading cause of infant hearing loss and neurodevelopmental delay, but vaccine candidates have faced challenges eliciting neutralizing antibodies. One of the most well-studied targets for CMV vaccines is the viral fusogen glycoprotein B (gB), which is required for viral entry into host cells. Within gB, antigenic domain 2 site 1 (AD-2S1) is a target of potently neutralizing antibodies, but gB-based candidate vaccines have yet to elicit robust responses against this region. We mapped the genealogy of B cells encoding potently neutralizing anti-gB AD-2S1 antibodies from their inferred unmutated common ancestor (UCA) and characterized the binding and function of early lineage ancestors. Remarkably, we found that the single amino acid heavy chain mutation A33N, an improbable mutation rarely generated by somatic hypermutation machinery, conferred broad CMV neutralization to the UCA antibody. Structural studies revealed that this mutation mediated key contacts with the gB AD-2S1 epitope. Collectively, these results provide insight into potently neutralizing gB-directed antibody evolution and a foundation for designing next-generation CMV vaccines. One-sentence summaryThis manuscript identifies an early B cell lineage mutation that confers neutralizing function to antibodies targeting CMV fusogen gB.

microbiology↗

bioRxiv2021

Zika virus-specific IgM elicited during pregnancy exhibits ultrapotent neutralization

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM responses contribute to ZIKV immunity in pregnancy, mediating neutralization up to three months post symptoms. From a ZIKV-infected pregnant woman, we established a B cell line secreting a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets a novel envelope dimer epitope within Domain II. The arrangement of this epitope on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not achievable by IgG antibodies. DH1017.IgM protected against lethal ZIKV challenge in mice. Our findings identify a unique role of antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunoprophylactic, particularly during pregnancy. Key pointsO_LIPlasma IgM contributes to early ZIKV neutralization during pregnancy C_LIO_LIUltrapotent neutralization by pentameric DH1017.IgM mAb depends on isotype C_LIO_LIDH1017.IgM can engage all binding sites concurrently through different angles of approach C_LIO_LIDH1017.IgM protects mice against lethal ZIKV challenge C_LI

immunology↗

bioRxiv2021

Vaccine-induced, high magnitude HIV Env-specific antibodies with Fc-mediated effector functions are insufficient to protect infant rhesus macaques against oral SHIV infection

Improved access to antiretroviral therapy and antenatal care have significantly reduced in-utero and peri-partum mother-to-child HIV transmission. However, as breastmilk transmission of HIV still occurs at an unacceptable rate there remains a need to develop an effective vaccine for the pediatric population. Previously, we compared different HIV vaccine strategies, intervals, and adjuvants in infant rhesus macaques to optimize the induction of HIV envelope (Env)-specific antibodies with Fc-mediated effector function. Here, we tested the efficacy of an optimized vaccine regimen against oral SHIV acquisition in infant macaques. One group of 12 animals was immunized with 1086.c gp120 protein adjuvanted with 3M-052 in stable emulsion and Modified Vaccinia Ankara (MVA) virus vector expressing 1086.c HIV Env, while the control group (n=12) was immunized only with empty MVA. The first vaccine dose was given within 10 days of birth and booster doses were administered at weeks 6 and 12. The vaccine regimen induced Env-specific plasma IgG antibodies capable of antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Beginning at week 15, infants were exposed orally to escalating doses of heterologous SHIV-1157(QNE)Y173H once a week until infected. Despite the induction of strong Fc-mediated antibody responses, the vaccine regimen did not reduce the risk of infection, time to acquisition, or peak viremia compared to controls. Our results suggest that the non-neutralizing Env-specific antibodies with Fc effector function elicited by this vaccine regimen were insufficient for protection against heterologous oral SHIV infection shortly after the final immunization. IMPORTANCEWomen of childbearing age are three times more likely to contract HIV infection than their male counterparts. Poor HIV testing rates coupled with low adherence to antiretroviral therapy (ART) result in a high risk of mother-to-infant HIV transmission, especially during the breastfeeding period. A preventative vaccine could curb pediatric HIV infections, reduce potential health sequalae, and prevent the need for lifelong ART in this population. The results of the current study imply that the HIV Env-specific IgG antibodies elicited by this candidate vaccine regimen, despite high magnitude of Fc-mediated effector function, but lack of neutralizing antibodies and polyfunctional T cell responses, were insufficient to protect infant rhesus macaques against oral virus acquisition.

immunology↗

bioRxiv2021

Protective transfer: Maternal passive immunization with a rotavirus-neutralizing dimeric IgA protects against rotavirus disease in suckling neonates

Breast milk secretory IgA antibodies provide a first line of defense against enteric infections. Despite this and an effective vaccine, human rotaviruses (RVs) remain the leading cause of severe infectious diarrhea in children in low- and middle-income countries (LMIC) where vaccine efficacy is lower than that of developed nations. Therapeutic strategies that deliver potently neutralizing antibodies into milk could provide protection against enteric pathogens such as RVs. We developed a murine model of maternal protective-transfer using systemic administration of a dimeric IgA (dIgA) monoclonal antibody. We confirmed that systemically-administered dIgA passively transferred into milk and stomach of suckling pups in a dose-dependent manner. We then demonstrated that systemic administration of an engineered potent RV-neutralizing dIgA (mAb41) in lactating dams protected suckling pups from RV-induced diarrhea. This maternal protective-transfer immunization platform could be an effective strategy to improve infant mortality against enteric infections, particularly in LMIC with high rates of breastfeeding. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/461116v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1af34dcorg.highwire.dtl.DTLVardef@1b78cb2org.highwire.dtl.DTLVardef@1760a62org.highwire.dtl.DTLVardef@a3cea2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Different adjuvanted pediatric HIV envelope vaccines induced distinct plasma antibody responses despite similar B cell receptor repertoires in infant rhesus macaques.

Different HIV vaccine regimens elicit distinct plasma antibody responses in both human and nonhuman primate models. Previous studies in human and non-human primate infants showed that adjuvants influenced the quality of plasma antibody responses induced by pediatric HIV envelope vaccine regimens. We recently reported that use of the 3M052-SE adjuvant and longer intervals between vaccinations are associated with higher magnitude of antibody responses in infant rhesus macaques. However, the impact of different adjuvants in HIV vaccine regimens on the developing infant B cell receptor (BCR) repertoire has not been studied. This study evaluated whether pediatric HIV envelope vaccine regimens with different adjuvants induced distinct antigen-specific memory B cell repertoires and whether specific immunoglobulin (Ig) immunogenetic characteristics are associated with higher magnitude of plasma antibody responses in vaccinated infant rhesus macaques. We utilized archived preclinical pediatric HIV vaccine studies PBMCs and tissue samples from 19 infant rhesus macaques immunized either with (i) HIV Env protein with a squalene adjuvant, (ii) MVA-HIV and Env protein coadministered using a 3-week interval, (iii) MVA-HIV prime/ protein boost with an extended 6-week interval between immunizations, or (iv) with HIV Env administered with 3M-052-SE adjuvant. Frequencies of vaccine-elicited HIV Env-specific memory B cells from PBMCs and tissues were similar across vaccination groups (frequency range of 0.06-1.72%). There was no association between vaccine-elicited antigen-specific memory B cell frequencies and plasma antibody titer or avidity. Moreover, the epitope specificity and Ig immunogenetic features of vaccine-elicited monoclonal antibodies did not differ between the different vaccine regimens. These data suggest that pediatric HIV envelope vaccine candidates with different adjuvants that previously induced higher magnitude and quality of plasma antibody responses in infant rhesus macaques were not driven by distinct antigen-specific memory BCR repertoires.

immunology↗

bioRxiv2021

Frequent development of broadly neutralizing antibodies in early life in a large cohort of HIV-infected children

Recent studies conducted in small cohorts of children have indicated that broadly neutralizing antibodies (bnAbs) may develop earlier after HIV infection compared to adults. To define the frequency and kinetics of bnAb responses in a larger pediatric cohort, we evaluated plasma from 212 ART-naive, children living with HIV aged 1 to 3 years. Neutralization breadth and potency was assessed using a panel of 10 tier-2 viruses and compared to those of adults with chronic HIV. Further, the magnitude, epitope specificity and IgG subclass distribution of Env-specific antibodies were also assessed using a binding antibody multiplex assay. We found that 1-year-old children demonstrated neutralization breadth comparable to that of chronically-infected adults, and breadth continued to increase with age such that the pediatric cohort overall exhibited significantly greater neutralization breadth than adults (p= 0.014). Similarly, binding antibody responses increased with age, and the levels in 2 to 3 year-old children were comparable to those of adults. Overall, there was no significant difference in antibody specificities or IgG subclass distribution between the pediatric and adult cohorts. Interestingly, the neutralization activity was mapped to a single epitope (CD4 binding site, V2 or V3 glycans) in only 5 of 38 pediatric broadly neutralizing samples, suggesting a polyclonal neutralization response may develop in most children. These results contribute to a growing body of evidence suggesting that the early life immune system may present advantages for the development of an effective HIV vaccine. Author summaryUnderstanding the development of broadly neutralizing antibodies during natural HIV infection is critical to provide insights for the development of an effective vaccine. In this study, we used a large cohort (n=212) of HIV infected children aged 1-3 years old to define the frequency and kinetics of broad neutralization in comparison to adults with chronic HIV. We also evaluated the magnitude and epitope specificity of HIV Env-specific antibodies in children and adults. We observed that both the binding and neutralizing antibody responses increased with age. However, by one year of age, children had neutralization breadth comparable to that of chronically infected adults; and overall, 1-3 year-old children demonstrated higher neutralization breadth than adults. Only slight differences in epitope-specificity and IgG subclass distribution were observed between children and adults, suggesting that these factors are not major contributors to the observed age-related difference in neutralization breadth. The epitope specificity of the neutralizing antibodies could not be mapped in the majority of children, suggesting that their response may be polyclonal. Our study contributes to the body of work indicating a potential advantage of the early life immune system for the elicitation of broad neutralization.

immunology↗

bioRxiv2021

SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

Early life SARS-CoV-2 vaccination has the potential to provide lifelong protection and achieve herd immunity. To evaluate SARS-CoV-2 infant vaccination, we immunized two groups of 8 infant rhesus macaques (RMs) at weeks 0 and 4 with stabilized prefusion SARS-CoV-2 S-2P spike (S) protein, either encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or mixed with 3M-052-SE, a TLR7/8 agonist in a squalene emulsion (Protein+3M-052-SE). Neither vaccine induced adverse effects. High magnitude S-binding IgG and neutralizing infectious dose 50 (ID50) >103 were elicited by both vaccines. S-specific T cell responses were dominated by IL-17, IFN-{gamma}, or TNF-. Antibody and cellular responses were stable through week 22. The S-2P mRNA-LNP and Protein-3M-052-SE vaccines are promising pediatric SARS-CoV-2 vaccine candidates to achieve durable protective immunity. One-Sentence SummarySARS-CoV-2 vaccines are well-tolerated and highly immunogenic in infant rhesus macaques

immunology↗

bioRxiv2021

Oral Hsp90 inhibitor, SNX-5422, attenuates SARS-CoV-2 replication and dampens inflammation in airway cells.

Currently available SARS-CoV-2 therapeutics are targeted towards moderately to severely ill patients and require intravenous infusions, with limited options for exposed or infected patients with no or mild symptoms. While vaccines have demonstrated protective efficacy, vaccine hesitancy and logistical distribution challenges will delay their ability to end the pandemic. Hence, there is a need for rapidly translatable, easy-to-administer-therapeutics, that can prevent SARS-CoV-2 disease progression, when administered in the early stages of infection. We demonstrate that an orally bioavailable Hsp90 inhibitor, SNX-5422, currently in clinical trials as an anti-cancer therapeutic, inhibits SARS-CoV-2 replication in vitro at a high selectivity index. SNX-5422 treatment of human primary airway epithelial cells dampened expression of inflammatory pathways associated with poor SARS-CoV-2 disease outcomes. Additionally, SNX-5422 interrupted expression of host factors that are crucial for SARS-CoV-2 replication machinery. Development of SNX-5422 as SARS-CoV-2-early-therapy will dampen disease severity, resulting in better clinical outcomes and reduced hospitalizations.

microbiology↗

bioRxiv2020

Specificity and effector functions of non-neutralizing gB-specific monoclonal antibodies isolated from healthy individuals with human cytomegalovirus infection

Human cytomegalovirus (HCMV) is the most common congenital infection, and the leading nongenetic cause of sensorineural hearing loss (SNHL) in newborns globally. A gB subunit vaccine administered with adjuvent MF59 (gB/MF59) is the most efficacious tested to-date, achieving 50% efficacy in preventing infection of HCMV-seronegative mothers. We recently discovered that gB/MF59 vaccination elicited primarily non-neutralizing antibody responses, that HCMV strains acquired by vaccinees more often included strains with gB genotypes that are distinct from the vaccine antigen, and that protection against HCMV acquisition correlated with ability of vaccine-elicited antibodies to bind to membrane associated gB. Thus, we hypothesized that gB-specific non-neutralizing antibody binding breadth and function are dependent on their epitope and genotype specificity as well as their ability to interact with membrane-associated gB. Twenty-four gB-specific monoclonal antibodies (mAbs) isolated from naturally HCMV-infected individuals were mapped for gB domain specificity by binding antibody multiplex assay (BAMA) and for genotype preference binding to membrane-associated gB presented on transfected cells. We defined their non-neutralizing functions including antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC). The isolated gB-specific non-neutralizing mAbs were primarily specific for Domain II and linear antigenic domain 2 site 2 (AD2). We observed variability in mAb gB genotype binding preference, with increased binding to gB genotypes 2 and 4. Functional studies identified two gB-specific mAbs that facilitate ADCP and have binding specificities of AD2 and Domain II. This investigation provides novel understanding on the impact of gB domain specificity and antigenic variability on gB-specific non-neutralizing antibody responses. ImportanceHCMV is the most common congenital infection worldwide, but development of a successful vaccine remains elusive. gB-specific non-neutralizing mAbs, represent a distinct anti-HCMV Ab subset implicated in the protection against primary infection during numerous phase-II gB/MF59 vaccine trials. By studying non-neutralizing gB-specific mAbs from naturally infected individuals, this study provides novel characterization of binding site specificity, genotypic preference, and effector cell functions mediated by mAbs elicited in natural infection. We found that a panel of twenty-four gB-specific non-neutralizing mAbs bind across multiple regions of the gB protein, traditionally through to be targeted by neutralizing mAbs only, and bind differently to gB depending if the protein is soluble versus embedded in a membrane. This investigation provides novel insight into the gB-specific binding characteristics and effector cell functions mediated by non-neutralizing gB-specific mAbs elicited through natural infection, providing new endpoints for future vaccine development.

immunology↗

bioRxiv2019

Maternal alum-adjuvanted recombinant HIV Env vaccine does not enhance autologous virus neutralization in HIV-infected pregnant women

Preventive strategies beyond ART will be required to end the pediatric HIV epidemic. A maternal vaccine capable of boosting neutralizing antibody (nAb) responses against circulating viruses in HIV-infected pregnant women could effectively decrease mother-to-child transmission of HIV. However, it is not known if an HIV envelope (Env) vaccine administered to infected pregnant women can enhance autologous virus neutralization. Here, we assessed autologous virus nAb responses in maternal plasma samples obtained from AIDS Vaccine Evaluation Group (AVEG) Protocols 104 and 102, historical Phase I safety and immunogenicity trials of recombinant HIV Env subunit vaccines in HIV-infected pregnant women (NCT00001041). AVEG 104 participants were randomized to receive 300 {micro}g Env subunit MN recombinant gp120 with alum adjuvant or alum alone. AVEG 102 participants were randomized to receive 640 {micro}g Env subunit recombinant gp160 or placebo. HIV Env-specific maternal plasma binding and neutralizing responses were characterized before and after vaccination in 15 AVEG 104 (n=10 vaccinee, n=5 placebo) and 2 AVEG 102 (n=1 vaccinee, n=1 placebo) participants. Single genome amplification (SGA) was used to obtain HIV env gene sequences from autologous viruses for pseudovirus production in pre- and post-vaccination plasma of HIV-infected pregnant vaccinees (n=6 gp120, n=1 gp160) and placebo recipients (n=3). We detected an increase in MN gp120-specific IgG binding in the vaccinee group between the first immunization visit and the last visit at delivery (p=0.027, 2-sided Wilcoxon test). However, no difference was observed in the neutralization potency of maternal plasma collected at delivery against autologous viruses isolated from early or late pregnancy. Thus, maternal vaccination with gp120/160 did not boost maternal autologous virus nAb responses. Immunization strategies capable of more potent B cell stimulation will likely be required to effectively boost autologous virus nAb responses in pregnant women and synergize with ART to further reduce infant HIV infections. HighlightsO_LIPrior maternal HIV Env vaccine trial did not assess autologous virus neutralization C_LIO_LICirculating viruses isolated from mothers were tested against autologous plasma C_LIO_LIMaternal vaccination with HIV Env gp120/160 increased MN gp120-specific IgG binding C_LIO_LIMaternal HIV Env vaccine regimen did not boost autologous virus neutralization C_LIO_LIMore potent B cell stimulation will be required to elicit autologous nAb responses C_LI

immunology↗

bioRxiv2019

Impact of preexisting virus-specific maternal antibodies on cytomegalovirus population genetics in a monkey model of congenital transmission

Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have investigated the genetic composition of viral populations in newborns diagnosed with HCMV, little is known regarding mother-to-child viral transmission dynamics and how therapeutic interventions may impact within-host viral populations. Here, we investigate how preexisting CMV-specific antibodies shape the maternal viral population and intrauterine virus transmission. Specifically, we characterize the genetic composition of CMV populations in a monkey model of congenital CMV infection to examine the effects of passively-infused hyperimmune globulin (HIG) on viral population genetics in both maternal and fetal compartments. In this study, 11 seronegative, pregnant monkeys were challenged with rhesus CMV (RhCMV), including a group pretreated with a standard potency HIG preparation (n = 3), a group pretreated with a high-neutralizing potency HIG preparation (n = 3), and an untreated control group (n = 5). Targeted amplicon deep sequencing of RhCMV glycoprotein B and L genes revealed that one of the three strains present in the viral inoculum (UCD52) dominated maternal and fetal viral populations. We identified de novo minor haplotypes of this strain and characterized their dynamics. Many of the identified haplotypes were consistently detected at multiple timepoints within sampled maternal tissues, as well as across tissue compartments, indicating haplotype persistence over time and transmission between maternal compartments. However, haplotype numbers and diversity levels were not appreciably different across HIG pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it has no apparent impact in the intrahost viral genetic diversity at the investigated loci. Interestingly, some haplotypes present in fetal and maternal-fetal interface tissues were also identified in maternal samples of corresponding dams, providing evidence for a wide RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies.\n\nAuthor summaryHuman cytomegalovirus (CMV) is the most common infectious cause of birth defects worldwide. Knowledge gaps remain regarding how maternal immunity impacts the genetic composition of CMV populations and the incidence of congenital virus transmission. Addressing these gaps is important to inform vaccine development efforts. Using viral samples collected from a monkey model of congenital CMV infection, we investigated the impact of passively-administered maternal antibodies on the genetic composition of the maternal virus population and that transmitted to the fetus. Our analysis focused on two CMV genes that encode glycoproteins that facilitate viral cellular entry and are known epitope targets of the humoral immune response. By identifying and analyzing variants across sampled maternal tissues, we found no impact in CMV genetic diversity by preexisting CMV-specific antibodies, despite the observation that such antibodies reduce viral load and confer some protection against congenital transmission. We further found that some minor variants identified in fetal and maternal-fetal interface tissues were also present in corresponding maternal tissues, indicating that a large number of viral particles passed from dam to fetus in observed cases of congenital transmission.

microbiology↗

bioRxiv2019

Analytical treatment interruption after short-term anti-retroviral therapy in a postnatally SHIV infected infant rhesus macaque model

To achieve long-term viral remission in HIV-infected children, novel strategies beyond early anti-retroviral therapy (ART) will be necessary. Identifying clinical predictors of time to viral rebound upon ART interruption will streamline the development of novel therapeutic strategies and accelerate their evaluation in clinical trials. However, identification of these biomarkers is logistically challenging in infants, due to sampling limitations and potential risks of treatment interruption. To facilitate identification of biomarkers predicting viral rebound, we have developed an infant rhesus macaque (RM) model of oral SHIV.CH505.375H.dCT challenge and analytical treatment interruption (ATI) after short-term ART. We used this model to characterize SHIV replication kinetics and virus-specific immune responses during short-term ART or post-ATI and demonstrated plasma viral rebound in 5 out of 6 (83%) infants. We observed a decline in humoral immune responses and partial dampening of systemic immune activation upon initiation of ART in these infants. Furthermore, we documented that infant and adult macaques have similar SHIV replication and rebound kinetics and equally potent virus-specific humoral immune responses. Finally, we validated our models by confirming a well-established correlate of time to viral rebound, namely pre-ART plasma viral load, as well as identified additional potential humoral immune correlates. Thus, this model of infant ART and viral rebound can be used and further optimized to define biomarkers of viral rebound following long-term ART as well as to pre-clinically assess novel therapies to achieve a pediatric HIV functional cure.\n\nIMPORTANCENovel interventions that do not rely on daily adherence to ART are needed to achieve sustained viral remission for perinatally infected children who currently rely on lifelong ART. Considering the risks and expense associated with ART-interruption trials, identification of biomarkers of viral rebound will prioritize promising therapeutic intervention strategies, including anti-HIV Env protein therapeutics. However, comprehensive studies to identify those biomarkers are logistically challenging in human infants, demanding the need for relevant non-human primate models of HIV rebound. In this study, we developed an infant RM model of oral Simian/Human Immunodeficiency virus infection expressing clade C HIV Env, and short-term ART followed by ATI, longitudinally characterizing immune responses to viral infection during ART and post-ATI. Additionally, we compared this infant RM model to an analogous adult RM rebound model and identified virologic and immunologic correlates of time to viral rebound post-ATI.

immunology↗

bioRxiv2019

Optimized Mucosal MVA Prime/ Soluble gp120 Boost Vaccination Regimen Induces Similar Antibody Responses as an Intramuscular Regimen

The benefits of mucosal vaccines over injected vaccines are difficult to ascertain since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia Ankara expressing HIV-1 gp120 (MVA-g120) prime and HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (IM) administered MVA prime/gp120 boost to allow comparison of an IM immunization regimen to a mucosal vaccination regimen for their ability to protect against a low dose rectal SHIV challenge while inducing similar serum anti-HIV-1 antibody responses. A 3-fold higher antigen dose was required for intranasal (IN) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by IM immunization. Gp120 fused to the Adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced IN immunogenicity when compared to gp120 alone. MVA-gp120 was more immunogenic after IN delivery than gastric or rectal delivery, although serum antibodies induced by IN immunization were lower than those induced by intramuscular immunization. Using these optimized vaccines, an IN MVA-gp120 prime, combined IM (gp120) and IN (gp120-Ad2F) boost regimen (IN/IM+IN) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (IM/IM) in rabbits and non-human primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the IM/IM nor the IN/IM+IN regimen induced elevated anti-HIV-1 mucosal IgA responses nor protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the IN route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunization\n\nIMPORTANCEMucosal vaccination is proposed as a method of immunization able to induce protection against mucosal pathogens that is superior to protection provided by parenteral immunization. However, mucosal vaccination often induces serum antigen-specific immune responses of lower magnitude than those induced by parenteral immunization, making the comparison of mucosal and parenteral immunization difficult. We identified vaccine parameters that allowed an immunization regimen consisting of an IN prime followed with boosters administered by both IN and IM routes to induce serum antibody responses similar to those induced by IM prime/boost vaccination. Additional studies are needed to determine the potential benefit of mucosal immunization for HIV-1 and other mucosally-transmitted pathogens.

immunology↗

bioRxiv2019

Oral clade C SHIV challenge models to study pediatric HIV-1 infection by breastmilk transmission

Nonhuman primate (NHP) models are invaluable for HIV pathogenesis, intervention and cure studies. To enhance the translational potential of NHP HIV vaccine studies, clinically relevant R5-tropic, tier 2 neutralization sensitive, and mucosally transmissible simian-human immunodeficiency viruses (SHIVs) have been designed. Towards our goal of developing vaccines to prevent breastmilk transmission of HIV, we evaluated virological outcomes of three distinct SHIVs in repeated weekly oral exposure regimens in infant rhesus macaques. The selected strains SHIV-1157ipd3N4, SHIV-1157(QNE)Y173H, and SHIV CH505 375H.dCT express a clade C HIV Env, the clade most prevalent in regions with high pediatric HIV infections. All three SHIVs were orally transmissible. However, compared to the pediatric SIVmac251 model, SHIV replication was more attenuated. Some animals exposed to weekly low-dose (20 TCID50) SHIV-1157ipd3N4 had transient viremia blips associated with lack or delayed seroconversion. Animals with acute viremia [≥]10,000 viral RNA copies/ml seroconverted. This finding was reminiscent of an earlier study suggesting to continue challenges until a threshold of [≥]10,000 viral RNA copies/ml is surpassed to achieve seroconversion, one criterion of HIV diagnosis. All animals exposed weekly with higher doses (>104 TCID50) of SHIV-1157(QNE)Y173H or SHIV CH505 375h.dCT developed persistent infection with high peak viremia and seroconverted. Chronic viremia varied widely in all three SHIV infection models. Thus, although R5 clade C SHIVs are excellent tools to study prevention of virus acquisition, secondary virological outcomes of vaccine efficacy (e.g. risk of infection per exposure, modulation of peak viremia, viral set point) will require careful consideration and validation in SHIV challenge models. IMPORTANCEThe development of an effective HIV vaccine remains a top priority towards the goal of reducing the number of new HIV infections. Studies in nonhuman primate models of HIV infection have been instrumental in the preclinical evaluation of candidate vaccines. To assess the role of HIV Env-specific antibodies with broadly neutralizing or Fc-mediated effector function in these NHP models, the development and optimization of novel SHIV challenge models is required. We evaluate three different clade C HIV Env SHIVs for infectivity of infant macaques by the oral route. Our results demonstrate that SHIV-1157ipd3N4, SHIV-1157(QNE)Y173H, and SHIV CH505 375H.dCT can be orally transmitted and establish persistent infection in infant rhesus macaques, but chronic viremia levels vary widely. Therefore, SHIV infection models are most pertinent when prevention of systemic infection is the primary readout of vaccine efficacy, but secondary outcome measures of vaccine efficacy will require stringent criteria and extensive validation.

microbiology↗

bioRxiv2019

SHIV.CH505-infected infant and adult rhesus macaques exhibit similar HIV Env-specific antibody kinetics, despite distinct T-follicular helper (Tfh) and germinal center B cell landscapes

Pediatric HIV infection remains a large global health concern despite the widespread use of antiretroviral therapy (ART). Thus, global elimination of pediatric HIV infections will require the development of novel immune-based approaches, and understanding infant immunity to HIV is critical to guide the rational design of these intervention strategies. Despite their immunological immaturity, HIV-infected children develop broadly neutralizing antibodies (bnAbs) more frequently and earlier than adults do. Furthermore, T-follicular helper (Tfh) cells have been associated with bnAb development in HIV-infected children and adults. To further our understanding of age-related differences in the development of HIV-specific immunity, we evaluated the generation of virus-specific humoral immune responses in infant (n=6) and adult (n=12) rhesus macaques (RMs) infected with a transmitted/founder (T/F) simian-human immunodeficiency virus (SHIV.C.CH505). The plasma HIV envelope-specific IgG antibody kinetics were similar in SHIV-infected infant and adult RMs, with no significant differences in the magnitude or breadth of these responses. Interestingly, autologous tier 2 virus neutralization responses also developed with similar frequency and kinetics in infant and adult RMs, despite infants exhibiting significantly higher Tfh and germinal center B cell frequencies compared to adults. Our results indicate that the humoral immune response to SHIV infection develops with similar kinetics among infant and adult RMs, suggesting that the early life immune system is equipped to respond to HIV-1 and promote the production of neutralizing HIV antibodies. ImportanceThere is a lack of understanding on how the maturation of the infant immune system influences immunity to HIV infection, or how these responses differ from those of adults. Improving our knowledge of infant HIV immunity will help guide antiviral intervention strategies that take advantage of the unique infant immune environment to successfully elicit protective immune responses. We utilized a rhesus macaque model of SHIV infection as a tool to distinguish the differences in HIV humoral immunity in infants versus adults. Here, we demonstrate that the kinetics and quality of the infant humoral immune response to HIV are highly comparable to that of adults during the early phase of infection, despite distinct differences in their Tfh responses, indicating that slightly different mechanisms may drive infant and adult humoral immunity.

immunology↗

bioRxiv2018

Genetic signatures of human cytomegalovirus variants acquired by seronegative glycoprotein B vaccinees

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, and a frequent cause of hearing loss or debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV-associated congenital disease is a public health priority. One potential strategy is vaccination of women of child-bearing age to prevent maternal HCMV acquisition during pregnancy. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating approximately 50% protection against HCMV infection of seronegative women in multiple phase 2 trials. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as multiple sequencing methodologies to interrogate the magnitude and genetic composition of HCMV populations infecting gB/MF59 vaccinees and placebo recipients. We identified several differences between the viral dynamics of acutely-infected vaccinees and placebo recipients. First, there was reduced magnitude viral shedding in the saliva of gB vaccinees. Additionally, employing a panel of tests for genetic compartmentalization, we noted tissue-specific gB haplotypes in the majority of vaccinees though only in a single placebo recipient. Finally, we observed reduced acquisition of genetically-related gB1, gB2, and gB4 genotype \"supergroup\" HCMV variants among vaccine recipients, suggesting that the gB1 genotype vaccine construct may have elicited partial protection against HCMV viruses with antigenically-similar gB sequences. These findings indicate that gB immunization may have had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.\n\nAuthor SummaryThough not a household name like Zika virus, human cytomegalovirus (HCMV) causes permanent neurologic disability in one newborn child every hour in the United States - more than Down syndrome, fetal alcohol syndrome, and neural tube defects combined. There are currently no established effective preventative measures to inhibit congenital HCMV transmission following acute or chronic HCMV infection of a pregnant mother. However, the glycoprotein B (gB) vaccine is the most effective HCMV vaccine tried clinically to date. Here, we utilized high-throughput, next-generation sequencing of viral DNA isolated from patients enrolled in a gB vaccine trial, and identified several impacts that this vaccine had on the size, distribution, and composition of the in vivo viral population. These results have increased our understanding of why the gB/MF59 vaccine was partially efficacious and will inform future rational design of a vaccine to prevent congenital HCMV.

microbiology↗

bioRxiv2018

HCMV glycoprotein B subunit vaccine efficacy was mediated by non-neutralizing antibody effector functions

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, frequently causing hearing loss and brain damage in afflicted infants. A vaccine to prevent maternal acquisition of HCMV during pregnancy is necessary to reduce the incidence of infant disease. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine platform is the most successful HCMV vaccine tested to-date, demonstrating approximately 50% efficacy in preventing HCMV acquisition in phase II trials. However, the mechanism of vaccine protection remains unknown. Plasma from 33 gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and non-neutralizing anti-HCMV effector functions, and compared to an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and Domain I. Interestingly, vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this non-neutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound trimeric, membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, though were poor mediators of NK cell activation. Altogether, these data suggest that non-neutralizing antibody functions, including virion phagocytosis, likely played a role in the observed 50% vaccine-mediated protection against HCMV acquisition.\n\nSignificanceThe CDC estimates that every hour, a child is born in the United States with permanent neurologic disability resulting from human cytomegalovirus (HCMV) infection - more than is caused by Down syndrome, fetal alcohol syndrome, and neural tube defects combined. A maternal vaccine to block transmission of HCMV to the developing fetus is a necessary intervention to prevent these adverse outcomes. The gB/MF59 vaccine is the most successful tested clinically to-date, achieving 50% reduction in HCMV acquisition. This manuscript establishes the function and epitope specificity of the humoral response stimulated by this vaccine that may explain the partial vaccine efficacy. Understanding the mechanism of gB/MF59-elicited protective immune responses will guide rational design and evaluation of the next generation of HCMV vaccines.

immunology↗

bioRxiv2017

Infant Transmitted/Founder HIV-1 Viruses from Peripartum Transmission are Neutralization Resistant to Paired Maternal Plasma

Despite extensive genetic diversity of HIV-1 in chronic infection, infant HIV-1 infection involves selective transmission of a single or few maternal virus variants. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env)-specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we isolated HIV-1 env genes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Moreover, infant T/F pseudoviruses were overall more neutralization resistant compared to maternal non-transmitted plasma variants (p=0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the spector that enhancement of this response at the end of pregnancy could further reduce infant HIV infection risk.\n\nAuthor SummaryMother to child transmission (MTCT) of HIV-1 can occur during pregnancy (in utero), at the time of delivery (peripartum) or by breastfeeding (postpartum). With the availability of anti-retroviral therapy (ART), rate of MTCT of HIV-1 have been significantly lowered. However, significant implementation challenges remains in resource-poor areas, making it difficult to eliminate pediatric HIV. An improved understanding of the viral population (escape variants from autologous neutralizing antibodies) that lead to infection of infants at time of transmission will help in designing immune interventions to reduce vertical HIV-1 transmission. Here, we selected 16 HIV-1-infected mother-infant pairs from WITS cohort (from pre anti-retroviral era), where infants became infected peripartum. HIV-1 env gene sequences were obtained by the single genome amplification method. The sensitivity of these infant Env pseudoviruses against paired maternal plasma and a panel of broadly neutralizing monoclonal antibodies (bNAbs) was analyzed. We demonstrated that the infant T/F viruses were more resistant against maternal plasma than non-transmitted maternal variants, but sensitive to most (bNAbs). Signature sequence analysis of infant T/F and non-transmitted maternal variants revealed the potential importance of V3 and MPER region for resistance against to paired maternal plasma. These findings provide insights for the design of maternal immunization strategies to enhance neutralizing antibodies that target V3 region of autologous virus populations, which could work synergistically with maternal ARVs to further reduce the rate of peripartum HIV-1 transmission.

molecular biology↗