Multivariate analysis of FcR-mediated NK cell functions identifies unique clustering among humans and rhesus macaques.

Rhesus macaques (RMs) are a common pre-clinical model used to test HIV vaccine efficacy and passive immunization strategies. Yet, it remains unclear to what extent the Fc-Fc receptor (FcR) interactions impacting antiviral activities of antibodies in RMs recapitulate those in humans. Here, we evaluated the FcR-related functionality of natural killer cells (NKs) from peripheral blood of uninfected humans and RMs to identify intra- and inter-species variation. NKs were screened for FcγRIIIa (human) and FcγRIII (RM) genotypes (FcγRIII(a)), receptor signaling, and antibody-dependent cellular cytotoxicity (ADCC), the latter mediated by a cocktail of monoclonal IgG1 antibodies with human or RM Fc. FcγRIII(a) genetic polymorphisms alone did not explain differences in NK effector functionality in either species cohort. Using the same parameters, hierarchical clustering separated each species into two clusters. Importantly, in principal components analyses, ADCC magnitude, NK contribution to ADCC, FcγRIII(a) cell-surface expression, and frequency of phosphorylated CD3ζ NK cells all contributed similarly to the first principal component within each species, demonstrating the importance of measuring multiple facets of NK cell function. Although ADCC potency was similar between species, we detected significant differences in frequencies of NK cells and pCD3ζ+ cells, level of cell-surface FcγRIII(a) expression, and NK-mediated ADCC (P<0.001), indicating that a combination of Fc-FcR parameters contribute to overall inter-species functional differences. These data strongly support the importance of multi-parameter analyses of Fc-FcR NK-mediated functions when evaluating efficacy of passive and active immunizations in pre- and clinical trials and identifying correlates of protection. The results also suggest that pre-screening animals for multiple FcR-mediated NK function would ensure even distribution of animals among treatment groups in future preclinical trials.

Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype.

Nearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, detection was not at the earliest stages of acute infection. Here, we identified an X4-tropic T/F HIV-1 in a participant in acute infection cohort. Coreceptor assays demonstrated that this T/F virus is strictly CXCR4 tropic. The participant experienced significantly faster CD4 depletion compared with R5 virus infected participants in the same cohort. Naïve and central memory CD4 subsets declined faster than effector and transitional memory subsets. All CD4 subsets, including naïve, were productively infected. Increased CD4+ T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions. These findings demonstrate that X4-tropic HIV-1 is transmissible through the mucosal route in people with the wild-type CCR5 genotype and have implications for understanding the transmissibility and immunopathogenesis of X4-tropic HIV-1.

AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo.

Persistent HIV-1 reservoirs of infected CD4 T cells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained in vivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 T cells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.

Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype.

Nearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, detection was not at the earliest stages of acute infection. Here, we identified an X4-tropic T/F HIV-1 in a participant in acute infection cohort. Coreceptor assays demonstrated that this T/F virus is strictly CXCR4 tropic. The participant experienced significantly faster CD4 depletion compared with R5 virus infected participants in the same cohort. Naïve and central memory CD4 subsets declined faster than effector and transitional memory subsets. All CD4 subsets, including naïve, were productively infected. Increased CD4 + T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions. These findings demonstrate that X4-tropic HIV-1 is transmissible through the mucosal route in people with the wild-type CCR5 genotype and have implications for understanding the transmissibility and immunopathogenesis of X4-tropic HIV-1.

Single-cell transcriptomics identifies prothymosin α restriction of HIV-1 in vivo.

Host restriction factors play key roles in innate antiviral defense, but it remains poorly understood which of them restricts HIV-1 in vivo. Here, we used single-cell transcriptomic analysis to identify host factors associated with HIV-1 control during acute infection by correlating host gene expression with viral RNA abundance within individual cells. Wide sequencing of cells from one participant with the highest plasma viral load revealed that intracellular viral RNA transcription correlates inversely with expression of the gene PTMA, which encodes prothymosin α. This association was genome-wide significant (Padjusted < 0.05) and was validated in 28 additional participants from Thailand and the Americas with HIV-1 CRF01_AE and subtype B infections, respectively. Overexpression of prothymosin α in vitro confirmed that this cellular factor inhibits HIV-1 transcription and infectious virus production. Our results identify prothymosin α as a host factor that restricts HIV-1 infection in vivo, which has implications for viral transmission and cure strategies.

Distinct mucosal and systemic immunological characteristics in transgender women potentially relating to HIV acquisition.

Transgender women (TGW) are disproportionally affected by HIV infection, with a global estimated prevalence of 19.9%, often attributed to behavioral risk factors, with less known about biological factors. We evaluated potential biological risk factors for HIV acquisition in TGW at the sites of viral entry by assessing immune parameters of the neovaginal surface and gut mucosa. The neovagina in TGW, compared with the vagina in cisgender women (CW), shows distinct cell composition and may pose a more inflammatory environment, evidenced by increased CD4+ T cell activation and higher levels of soluble markers of inflammation (C-reactive protein, soluble CD30). Increased inflammation may be driven by microbiome composition, as shown by a greater abundance of Prevotella and a higher Shannon Diversity Index. In addition, we have observed higher frequency of CD4+CCR5+ target cells and decreased DNA methylation of the CCR5 gene in the gut mucosa of TGW compared with CW and men who have sex with men, which was inversely correlated with testosterone levels. The rectal microbiome composition in TGW appears to favor a proinflammatory milieu as well as mucosal barrier disruption. Thus, it is possible that increased inflammation and higher frequencies of CCR5-expressing target cells at sites of mucosal viral entry may contribute to increased risk of HIV acquisition in TGW, with further validation in larger studies warranted.

HLA-B*57 and B*58 Associate with Predictors of Reservoir Size in an Acutely Treated HIV Cohort.

Much has been learnt about the role of human leukocyte antigen (HLA) alleles during natural infection of HIV-1, but far less is known about their role in people living with HIV (PLWH) on suppressive antiretroviral therapy (ART). In this study we used variable selection to identify predictors of HIV reservoir size, as measured by total HIV DNA in 192 participants in an acute HIV infection (AHI) cohort. Baseline clinical data including pre-ART CD4 T cell counts and plasma viral load (VL) were available from all participants along with longitudinal measurements after ART initiation during AHI. Time to VL suppression, time to CD4 reconstitution, and pre-ART viremia were the strongest predictors of undetectable total HIV DNA at 24 weeks after ART initiation. We next performed HLA typing in 526 participants from the same cohort and investigated associations with the three predictors of reservoir size. HLA-B*57 and B*58 both associated significantly with time to VL suppression, which was one of the predictors of the size of the HIV reservoir. These findings are significant in PLWH and have to be considered in the context of therapeutic intervention when conducting analytic treatment interruption studies as participants with these alleles could impact clinical findings given the small sizes of these studies.

Fc receptor engagement of HIV-1 Env-specific antibodies in mothers and infants predicts reduced vertical transmission.

Introduction: Infants acquire maternal antibodies by Fc receptor transcytosis across the placenta during pregnancy. Fc receptors are expressed on immune cells and are important for activation of effector cell functions. Methods: In this study, we evaluated Fc receptor engagement and ADCC activity of plasma binding antibodies from human immunodeficiency virus-1 (HIV) -infected mothers and to identify factors that may contribute to protection from HIV vertical transmission. Results: HIV-specific binding and Fc receptor engagement of plasma antibodies varied between mothers by transmission status and infants by infection status. Non-transmitting (NT) mothers and HIV-uninfected infants had antibodies with higher neonatal Fc receptor (FcRn) and FcγR engagement, as compared to transmitting (T) mothers and HIV+ infants, respectively. A significant inverse correlation between plasma antibody FcRn and FcγR engagement was observed for T mothers, but not NT mothers. Conversely, a significant direct correlation was observed between plasma antibody FcRn and FcγR engagement for HIV- infants, but not for HIV+ infants. Consequently, we observed significantly higher plasma antibody ADCC potency and breadth in HIV- infants, as compared to HIV+ infants. However, no differences in overall ADCC potency and breadth were observed between mothers. FcRn-engagement of HIV-specific antibodies in both mothers and infants predicted a lack of vertical transmission of HIV. Discussion: This study indicates that HIV-uninfected infants acquire HIV-specific antibodies with greater Fc receptor engagement and thus, greater ADCC capacity.

Long-term antiretroviral therapy initiated in acute HIV infection prevents residual dysfunction of HIV-specific CD8+ T cells.

BACKGROUND: Harnessing CD8+ T cell responses is being explored to achieve HIV remission. Although HIV-specific CD8+ T cells become dysfunctional without treatment, antiretroviral therapy (ART) partially restores their function. However, the extent of this recovery under long-term ART is less understood. METHODS: We analyzed the differentiation status and function of HIV-specific CD8+ T cells after long-term ART initiated in acute or chronic HIV infection ex vivo and upon in vitro recall. FINDINGS: ART initiation in any stage of acute HIV infection promoted the persistence of long-lived HIV-specific CD8+ T cells with high expansion (P<0·0008) and cytotoxic capacity (P=0·02) after in vitro recall, albeit at low cell number (P=0·003). This superior expansion capacity correlated with stemness (r=0·90, P=0·006), measured by TCF-1 expression, similar to functional HIV-specific CD8+ T cells found in spontaneous controllers. Importanly, TCF-1 expression in these cells was associated with longer time to viral rebound ranging from 13 to 48 days after ART interruption (r =0·71, P=0·03). In contrast, ART initiation in chronic HIV infection led to more differentiated HIV-specific CD8+ T cells lacking stemness properties and exhibiting residual dysfunction upon recall, with reduced proliferation and cytolytic activity. INTERPRETATION: ART initiation in acute HIV infection preserves functional HIV-specific CD8+ T cells, albeit at numbers too low to control viral rebound post-ART. HIV remission strategies may need to boost HIV-specific CD8+ T cell numbers and induce stem cell-like properties to reverse the residual dysfunction persisting on ART in people treated after acute infection prior to ART release. FUNDING: U.S. National Institutes of Health and U.S. Department of Defense.

Genetic variation that determines TAPBP expression levels associates with the course of malaria in an HLA allotype-dependent manner.

HLA class I (HLA-I) allotypes vary widely in their dependence on tapasin (TAPBP), an integral component of the peptide-loading complex, to present peptides on the cell surface. We identified two single-nucleotide polymorphisms that regulate TAPBP messenger RNA (mRNA) expression in Africans, rs111686073 (G/C) and rs59097151 (A/G), located in an AP-2α transcription factor binding site and a microRNA (miR)-4486 binding site, respectively. rs111686073G and rs59097151A induced significantly higher TAPBP mRNA expression relative to the alternative alleles due to higher affinity for AP-2α and abrogation of miR-4486 binding, respectively. These variants associated with lower Plasmodium falciparum parasite prevalence and lower incidence of clinical malaria specifically among individuals carrying tapasin-dependent HLA-I allotypes, presumably by augmenting peptide loading, whereas tapasin-independent allotypes associated with relative protection, regardless of imputed TAPBP mRNA expression levels. Thus, an attenuated course of malaria may occur through enhanced breadth and/or magnitude of antigen presentation, an important consideration when evaluating vaccine efficacy.

Acute HIV-1 infection viremia associate with rebound upon treatment interruption.

BACKGROUND: Analytic treatment interruption (ATI) studies evaluate strategies to potentially induce remission in people living with HIV-1 but are often limited in sample size. We combined data from four studies that tested three interventions (vorinostat/hydroxychloroquine/maraviroc before ATI, Ad26/MVA vaccination before ATI, and VRC01 antibody infusion during ATI). METHODS: The statistical validity of combining data from these participants was evaluated. Eleven variables, including HIV-1 viral load at diagnosis, Fiebig stage, and CD4+ T cell count were evaluated using pairwise correlations, statistical tests, and Cox survival models. FINDINGS: Participants had homogeneous demographic and clinical characteristics. Because an antiviral effect was seen in participants who received VRC01 infusion post-ATI, these participants were excluded from the analysis, permitting a pooled analysis of 53 participants. Time to viral rebound was significantly associated with variables measured at the beginning of infection: pre-antiretroviral therapy (ART) viral load (HR = 1.34, p = 0.022), time to viral suppression post-ART initiation (HR = 1.07, p < 0.001), and area under the viral load curve (HR = 1.34, p = 0.026). CONCLUSIONS: We show that higher viral loads in acute HIV-1 infection were associated with faster viral rebound, demonstrating that the initial stage of HIV-1 infection before ART initiation has a strong impact on viral rebound post-ATI years later. FUNDING: This work was supported by a cooperative agreement between the Henry M. Jackson Foundation for the Advancement of Military Medicine and the US Department of the Army (W81XWH-18-2-0040). This research was funded, in part, by the US National Institute of Allergy and Infectious Diseases (AAI20052001) and the I4C Martin Delaney Collaboratory (5UM1AI126603-05).

HLA-B∗46 associates with rapid HIV disease progression in Asian cohorts and prominent differences in NK cell phenotype.

Human leukocyte antigen (HLA) alleles have been linked to HIV disease progression and attributed to differences in cytotoxic T lymphocyte (CTL) epitope representation. These findings are largely based on treatment-naive individuals of European and African ancestry. We assessed HLA associations with HIV-1 outcomes in 1,318 individuals from Thailand and found HLA-B∗46:01 (B∗46) associated with accelerated disease in three independent cohorts. B∗46 had no detectable effect on HIV-specific T cell responses, but this allele is unusual in containing an HLA-C epitope that binds inhibitory receptors on natural killer (NK) cells. Unbiased transcriptomic screens showed increased NK cell activation in people with HIV, without B∗46, and simultaneous single-cell profiling of surface proteins and transcriptomes revealed a NK cell subset primed for increased responses in the absence of B∗46. These findings support a role for NK cells in HIV pathogenesis, revealed by the unique properties of the B∗46 allele common only in Asia.

Associations of human leukocyte antigen with neutralizing antibody titers in a tetravalent dengue vaccine phase 2 efficacy trial in Thailand.

The recombinant, live, attenuated, tetravalent dengue vaccine CYD-TDV has shown efficacy against all four dengue serotypes. In this exploratory study (CYD59, NCT02827162), we evaluated potential associations of host human leukocyte antigen (HLA) alleles with dengue antibody responses, CYD-TDV vaccine efficacy, and virologically-confirmed dengue (VCD) cases. Children 4-11 years old, who previously completed a phase 2b efficacy study of CYD-TDV in a single center in Thailand, were included in the study. Genotyping of HLA class I and II loci was performed by next-generation sequencing from DNA obtained from 335 saliva samples. Dengue neutralizing antibody titers (NAb) were assessed as a correlate of risk and protection. Regression analyses were used to assess associations between HLA alleles and NAb responses, vaccine efficacy, and dengue outcomes. Month 13 NAb log geometric mean titers (GMTs) were associated with decreased risk of VCD. In the vaccine group, HLA-DRB1*11 was significantly associated with higher NAb log GMT levels (beta: 0.76; p = 0.002, q = 0.13). Additionally, in the absence of vaccination, HLA associations were observed between the presence of DPB1*03:01 and increased NAb log GMT levels (beta: 1.24; p = 0.005, q = 0.17), and between DPB1*05:01 and reduced NAb log GMT levels (beta: -1.1; p = 0.001, q = 0.07). This study suggests associations of HLA alleles with NAb titers in the context of dengue outcomes. This study was registered with clinicaltrials.gov: NCT02827162.

Monocyte-derived transcriptome signature indicates antibody-dependent cellular phagocytosis as a potential mechanism of vaccine-induced protection against HIV-1.

A gene signature was previously found to be correlated with mosaic adenovirus 26 vaccine protection in simian immunodeficiency virus and simian-human immunodeficiency virus challenge models in non-human primates. In this report, we investigated the presence of this signature as a correlate of reduced risk in human clinical trials and potential mechanisms of protection. The absence of this gene signature in the DNA/rAd5 human vaccine trial, which did not show efficacy, strengthens our hypothesis that this signature is only enriched in studies that demonstrated protection. This gene signature was enriched in the partially effective RV144 human trial that administered the ALVAC/protein vaccine, and we find that the signature associates with both decreased risk of HIV-1 acquisition and increased vaccine efficacy (VE). Total RNA-seq in a clinical trial that used the same vaccine regimen as the RV144 HIV vaccine implicated antibody-dependent cellular phagocytosis (ADCP) as a potential mechanism of vaccine protection. CITE-seq profiling of 53 surface markers and transcriptomes of 53,777 single cells from the same trial showed that genes in this signature were primarily expressed in cells belonging to the myeloid lineage, including monocytes, which are major effector cells for ADCP. The consistent association of this transcriptome signature with VE represents a tool both to identify potential mechanisms, as with ADCP here, and to screen novel approaches to accelerate the development of new vaccine candidates.

High-resolution HLA allele and haplotype frequencies in several unrelated populations determined by next generation sequencing: 17th International HLA and Immunogenetics Workshop joint report.

The primary goal of the unrelated population HLA diversity (UPHD) component of the 17th International HLA and Immunogenetics Workshop was to characterize HLA alleles at maximum allelic-resolution in worldwide populations and re-evaluate patterns of HLA diversity across populations. The UPHD project included HLA genotype and sequence data, generated by various next-generation sequencing methods, from 4,240 individuals collated from 12 different countries. Population data included well-defined large datasets from the USA and smaller samples from Europe, Australia, and Western Asia. Allele and haplotype frequencies varied across populations from distant geographical regions. HLA genetic diversity estimated at 2- and 4-field allelic resolution revealed that diversity at the majority of loci, particularly for European-descent populations, was lower at the 2-field resolution. Several common alleles with identical protein sequences differing only by intronic substitutions were found in distinct haplotypes, revealing a more detailed characterization of linkage between variants within the HLA region. The examination of coding and non-coding nucleotide variation revealed many examples in which almost complete biunivocal relations between common alleles at different loci were observed resulting in higher linkage disequilibrium. Our reference data of HLA profiles characterized at maximum resolution from many populations is useful for anthropological studies, unrelated donor searches, transplantation, and disease association studies.

B cell engagement with HIV-1 founder virus envelope predicts development of broadly neutralizing antibodies.

Determining which immunological mechanisms contribute to the development of broad neutralizing antibodies (bNAbs) during HIV-1 infection is a major goal to inform vaccine design. Using samples from a longitudinal HIV-1 acute infection cohort, we found key B cell determinants within the first 14-43 days of viremia that predict the development of bNAbs years later. Individuals who develop neutralization breadth had significantly higher B cell engagement with the autologous founder HIV envelope (Env) within 1 month of initial viremia. A higher frequency of founder-Env-specific naive B cells was associated with increased B cell activation and differentiation and predictive of bNAb development. These data demonstrate that the initial B cell interaction with the founder HIV Env is important for the development of broadly neutralizing antibodies and provide evidence that events within HIV acute infection lead to downstream functional outcomes.

Dendritic cells focus CTL responses toward highly conserved and topologically important HIV-1 epitopes.

BACKGROUND: During early HIV-1 infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV-1 epitopes in HIV-1-infected individuals from the Thailand RV254/SEARCH 010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). METHODS: Autologous MDC1 were used as antigen presenting cells to induce in vitro CTL responses against HIV-1 Gag, Pol, Env, and Nef as determined by flow cytometry and ELISpot assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome. FINDINGS: MDC1 presenting either the overlapping Gag, Epigraph, or Network 14-21mer peptide pools consistently activated and expanded HIV-1-specific T cells to epitopes identified at the 9-13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens but a higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically constrained Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures. INTERPRETATION: Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward putative fitness-constrained regions of HIV-1 to prevent immune escape and control HIV-1 infection. FUNDING: A full list of the funding sources is detailed in the Acknowledgment section of the manuscript.

Next-generation sequencing of 11 HLA loci in a large dengue vaccine cohort from the Philippines.

HLA genotyping by next-generation sequencing (NGS) has evolved with significant advancements in the last decade. Here we describe full-length HLA genotyping of 11 loci in 612 individuals comprising a dengue vaccine cohort from Cebu province in the Philippines. The multi-locus individual tagging NGS (MIT-NGS) method that we developed initially for genotyping 4-6 loci in one MiSeq run was expanded to 11 loci including HLA-A, B, C, DPA1, DPB1, DQA1, DQB1, DRB1, and DRB3/4/5. This change did not affect the overall coverage or depth of the sequencing reads. HLA alleles with frequencies greater than 10% were A*11:01:01, A*24:02:01, A*24:07:01, A*34:01:01, B*38:02:01, B*15:35, B*35:05:01, C*07:02:01, C*04:01:01, DPA1*02:02:02, DPB1*05:01:01, DPB1*01:01:01, DQA1*01:02:01, DQA1*06:01:01, DQB1*05:02:01, DQB1*03:01:01, DRB1*15:02:01, DRB1*12:02:01, DRB3*03:01:03, DRB4*01:03:01, and DRB5*01:01:01. Improvements in sequencing library preparation provide uniform and even coverage across all exons and introns. This has led to a marked reduction in allele imbalance and dropout. Furthermore, including more loci, such as DRB3/4/5, decreases cross-mapping and incorrect allele assignment at the DRB1 locus. The increased number of loci sequenced for each sample does not reduce the number of samples that can be multiplexed on a single MiSeq run and is therefore more cost-efficient. We believe that such improvements will help HLA genotyping by NGS to gain momentum over other conventional methods by increasing confidence in the calls.

Safety and immunogenicity of Ad26 and MVA vaccines in acutely treated HIV and effect on viral rebound after antiretroviral therapy interruption.

We administered Ad26, modified vaccinia Ankara vectors containing mosaic HIV-1 antigens or placebo in 26 individuals who initiated antiretroviral therapy during acute human immunodeficiency virus infection as an exploratory study to determine the safety and duration of viremic control after treatment interruption. The vaccine was safe and generated robust immune responses, but delayed time to viral rebound compared to that in placebo recipients by only several days and did not lead to viremic control after treatment interruption (clinical trial NCT02919306).

Potent Zika and dengue cross-neutralizing antibodies induced by Zika vaccination in a dengue-experienced donor.

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. 1-3). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV)4-7 in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.