Adeno-associated viruses (AAVs) are popular gene therapy delivery vectors, but their application can be limited by anti-vector immunity. Both preexisting neutralizing antibodies (NAbs) and post-administration NAbs can limit transgene expression and reduce the clinical utility of AAVs. The development of novel AAVs will advance our understanding of AAV immunity and may also have practical applications. In this study, we identified five novel AAV capsids from rhesus macaques. RhAAV4282 exhibited 91.4% capsid sequence similarity with AAV7 and showed similar tissue tropism with slightly diminished overall signal. Despite this sequence homology, RhAAV4282 and AAV7 showed limited cross-neutralization. We determined a cryo-EM structure of the RhAAV4282 capsid at 2.57 Å resolution and identified a small segment within the hypervariable region IV, involving seven amino acids that formed a shortened external loop in RhAAV4282 compared with AAV7. We generated RhAAV4282 and AAV7 mutants that involved swaps of this region and showed that this region partially determined neutralization phenotype. We termed this region the hypervariable region IV neutralizing epitope (HRNE). Our data suggests that modification of the HRNE can lead to AAVs with altered neutralization profiles.

At the start of the Zika virus (ZIKV) epidemic in 2015, ZIKV spread across South and Central America, and reached parts of the southern United States placing pregnant women at risk for fetal microcephaly, fetal loss, and other adverse pregnancy outcomes associated with congenital ZIKA syndrome (CZS). For this reason, testing of a safe and efficacious ZIKV vaccine remains a global health priority. Here we report that a single immunization with Ad26.M.Env ZIKV vaccine, when administered prior to conception, fully protects pregnant rhesus macaques from ZIKV viral RNA in blood and tissues with no adverse effects in dams and fetuses. Furthermore, vaccination prevents ZIKV distribution to fetal tissues including the brain. ZIKV associated neuropathology was absent in offspring of Ad26.M.Env vaccinated dams, although pathology was limited in fetuses from non-immunized, challenged dams. Vaccine efficacy is associated with induction of ZIKV neutralizing antibodies in pregnant rhesus macaques. These data suggest the feasibility of vaccine prevention of CZS in humans.

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.

HIV-1 therapy with single or dual broadly neutralizing antibodies (bNAbs) has shown viral escape, indicating that at least a triple bNAb therapy may be needed for robust suppression of viremia. We performed a two-part study consisting of a single-center, randomized, double-blind, dose-escalation, placebo-controlled first-in-human trial of the HIV-1 V2-glycan-specific antibody PGDM1400 alone or in combination with the V3-glycan-specific antibody PGT121 in 24 adults without HIV in part 1, as well as a multi-center, open-label trial of the combination of PGDM1400, PGT121 and the CD4-binding-site antibody VRC07-523LS in five viremic adults living with HIV not on antiretroviral therapy (ART) in part 2 ( NCT03205917 ). The primary endpoints were safety, tolerability and pharmacokinetics for both parts and antiviral activity among viremic adults living with HIV and not on ART for part 2 of the study. The secondary endpoints were changes in CD4 + T cell counts and development of HIV-1 sequence variations associated with PGDM1400, PGT121 and VRC07-523LS resistance in part 2. Intravenously administered PGDM1400 was safe and well-tolerated at doses up to 30 mg kg −1 and when given in combination with PGT121 and VRC07-523LS. A single intravenous infusion of 20 mg kg −1 of each of the three antibodies reduced plasma HIV RNA levels in viremic individuals by a maximum mean of 2.04 log 10 copies per ml; however, viral rebound occurred in all participants within a median of 20 days after nadir. Rebound viruses demonstrated partial to complete resistance to PGDM1400 and PGT121 in vitro, whereas susceptibility to VRC07-523LS was preserved. Viral rebound occurred despite mean VRC07-523LS serum concentrations of 93 µg ml −1 . The trial met the pre-specified endpoints. Our data suggest that future bNAb combinations likely need to achieve broad antiviral activity, while also maintaining high serum concentrations, to mediate viral control.

The establishment of a long-lived viral reservoir is the key obstacle for achieving an HIV-1 cure. However, the anatomic, virologic, and immunologic features of the viral reservoir in tissues during antiretroviral therapy (ART) remain poorly understood. Here we present a comprehensive necroscopic analysis of the SIV/SHIV viral reservoir in multiple lymphoid and non-lymphoid tissues from SIV/SHIV-infected rhesus macaques suppressed with ART for one year. Viral DNA is observed broadly in multiple tissues and is comparable in animals that had initiated ART at week 1 or week 52 of infection. In contrast, viral RNA is restricted primarily to lymph nodes. Ongoing viral RNA transcription is not the result of unsuppressed viral replication, as single-genome amplification and subsequent phylogenetic analysis do not show evidence of viral evolution. Gag-specific CD8+ T cell responses are predominantly observed in secondary lymphoid organs in animals chronically infected prior to ART and these responses are dominated by CD69+ populations. Overall, we observe that the viral reservoir in rhesus macaques is widely distributed across multiple tissue sites and that lymphoid tissues act as a site of persistent viral RNA transcription under conditions of long-term ART suppression.

Human immunodeficiency virus (HIV)-1-specific broadly neutralizing monoclonal antibodies are currently under development to treat and prevent HIV-1 infection. We performed a single-center, randomized, double-blind, dose-escalation, placebo-controlled trial of a single administration of the HIV-1 V3-glycan-specific antibody PGT121 at 3, 10 and 30 mg kg –1 in HIV-uninfected adults and HIV-infected adults on antiretroviral therapy (ART), as well as a multicenter, open-label trial of one infusion of PGT121 at 30 mg kg –1 in viremic HIV-infected adults not on ART (no. NCT02960581). The primary endpoints were safety and tolerability, pharmacokinetics (PK) and antiviral activity in viremic HIV-infected adults not on ART. The secondary endpoints were changes in anti-PGT121 antibody titers and CD4 + T-cell count, and development of HIV-1 sequence variations associated with PGT121 resistance. Among 48 participants enrolled, no treatment-related serious adverse events, potential immune-mediated diseases or Grade 3 or higher adverse events were reported. The most common reactions among PGT121 recipients were intravenous/injection site tenderness, pain and headache. Absolute and relative CD4 + T-cell counts did not change following PGT121 infusion in HIV-infected participants. Neutralizing anti-drug antibodies were not elicited. PGT121 reduced plasma HIV RNA levels by a median of 1.77 log in viremic participants, with a viral load nadir at a median of 8.5 days. Two individuals with low baseline viral loads experienced ART-free viral suppression for ≥168 days following antibody infusion, and rebound viruses in these individuals demonstrated full or partial PGT121 sensitivity. The trial met the prespecified endpoints. These data suggest that further investigation of the potential of antibody-based therapeutic strategies for long-term suppression of HIV is warranted, including in individuals off ART and with low viral load.

Pre-existing immunity to flaviviruses can influence the outcome of subsequent flavivirus infections. Therefore, it is critical to determine whether baseline DENV immunity may influence subsequent ZIKV infection and the protective efficacy of ZIKV vaccines. In this study, we investigated the impact of pre-existing DENV immunity induced by vaccination on ZIKV infection and the protective efficacy of an inactivated ZIKV vaccine. Rhesus macaques and mice inoculated with a live attenuated DENV vaccine developed neutralizing antibodies (NAbs) to multiple DENV serotypes but no cross-reactive NAbs responses to ZIKV. Animals with baseline DENV NAbs did not exhibit enhanced ZIKV infection and showed no overall reduction in ZIKV vaccine protection. Moreover, passive transfer of purified DENV-specific IgG from convalescent human donors did not augment ZIKV infection in STAT2 -/- and BALB/c mice. In summary, these results suggest that baseline DENV immunity induced by vaccination does not significantly enhance ZIKV infection or impair the protective efficacy of candidate ZIKV vaccines in these models. These data can help inform immunization strategies in regions of the world with multiple circulating pathogenic flaviviruses.

Background: Zika virus (ZIKV) may cause severe congenital disease after maternal-fetal transmission. No vaccine is currently available. Objective: To assess the safety and immunogenicity of Ad26.ZIKV.001, a prophylactic ZIKV vaccine candidate. Design: Phase 1 randomized, double-blind, placebo-controlled clinical study. (ClinicalTrials.gov: NCT03356561) Setting: United States. Participants: 100 healthy adult volunteers. Intervention: Ad26.ZIKV.001, an adenovirus serotype 26 vector encoding ZIKV M-Env, administered in 1- or 2-dose regimens of 5 x 10(10) or 1 x 10(11) viral particles (vp), or placebo. Measurements: Local and systemic adverse events; neutralization titers by microneutralization assay (MN50) and T-cell responses by interferon-g enzyme-linked immunospot and intracellular cytokine staining; and protectivity of vaccine-induced antibodies in a subset of participants through transfer in an exploratory mouse ZIKV challenge model. Results: All regimens were well tolerated, with no safety concerns identified. In both 2-dose regimens, ZIKV neutralizing titers peaked 14 days after the second vaccination, with geometric mean MN50 titers (GMTs) of 1065.6 (95% CI, 494.9 to 2294.5) for 5 x 10(10) vp and 956.6 (595.8 to 1535.8) for 1 x 10(11) vp. Titers persisted for at least 1 year at a GMT of 68.7 (CI, 26.4-178.9) for 5 x 10(10) vp and 87.0 (CI, 29.3 to 258.6) for 1 x 10(11) vp. A 1-dose regimen of 1 x 10(11) vp Ad26.ZIKV.001 induced seroconversion in all participants 56 days after the first vaccination (GMT, 103.4 [CI, 52.7 to 202.9]), with titers persisting for at least 1 year (GMT, 90.2 [CI, 38.4 to 212.2]). Env-specific cellular responses were induced. Protection against ZIKV challenge was observed after antibody transfer from participants into mice, and MN50 titers correlated with protection in this model. Limitation: The study was conducted in a nonendemic area, so it did not assess safety and immunogenicity in a flavivirus-exposed population. Conclusion: The safety and immunogenicity profile makes Ad26.ZIKV.001 a promising candidate for further development if the need reemerges.

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. Zika virus vaccination elicits Zika and dengue virus cross-neutralizing antibodies in flavivirus-exposed individuals, potentially enhancing the protective efficacy of the vaccine in flavivirus-endemic regions.

The emergence and re-emergence of highly virulent viral pathogens with the potential to cause a pandemic creates an urgent need for the accelerated discovery of antiviral therapeutics. Antiviral human monoclonal antibodies (mAbs) are promising candidates for the prevention and treatment of severe viral diseases, but their long development timeframes limit their rapid deployment and use. Here, we report the development of an integrated sequence of technologies, including single-cell mRNA-sequence analysis, bioinformatics, synthetic biology and high-throughput functional analysis, that enables the rapid discovery of highly potent antiviral human mAbs, the activity of which we validated in vivo. In a 78-d study modelling the deployment of a rapid response to an outbreak, we isolated more than 100 human mAbs that are specific to Zika virus, assessed their function, identified that 29 of these mAbs have broadly neutralizing activity, and verified the therapeutic potency of the lead candidates in mice and non-human primate models of infection through the delivery of an antibody-encoding mRNA formulation and of the respective IgG antibody. The pipeline provides a roadmap for rapid antibody-discovery programmes against viral pathogens of global concern.

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV.

PROBLEM:Evaluation of Zika virus (ZIKV)-specific humoral and cellular immune response in pregnant women exposed to ZIKV.METHOD OF STUDY:In this observational, prospective cohort study, we recruited pregnant women presenting for prenatal ultrasound for ZIKV exposure at a single academic teaching hospital in Boston, MA from November 2016 to December 2018. We collected blood, urine, and cervicovaginal swabs antepartum, intrapartum, and postpartum; and cord blood and placenta at delivery. We used experimental assays to calculate quantitative viral loads, ZIKV-specific immunoglobulin titers, and ZIKV-specific T-cell responses.RESULTS:We enrolled 22 participants, three of which had serologic-confirmed ZIKV infection. No participants demonstrated sustained ZIKV shedding. ZIKV-specific IgG/IgM antibody was sustained throughout pregnancy and postpartum. ZIKV envelope and capsid-specific T-cell responses were also observed, albeit inconsistent. No newborns in this cohort had congenital Zika syndrome. Infant cord blood of infected mothers exhibited ZIKV-specific IgG, but not IgM antibodies.CONCLUSION:We detected a robust, prolonged maternal humoral immune response to ZIKV during pregnancy and postpartum. We also demonstrated evidence for efficient transplacental antibody transfer from mother to infant at birth, supporting the importance of neonatal passive immunity to ZIKV. Maternal T-cell responses were less consistent among pregnant women infected with ZIKV.

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization.

Viral rebound following antiretroviral therapy (ART) discontinuation in HIV-1-infected individuals is believed to originate from a small pool of CD4+ T cells harboring replication-competent provirus. However, the origin and nature of the rebound virus has remained unclear. Recent studies have suggested that rebound virus does not originate directly from individual latent proviruses but rather from recombination events involving multiple proviruses. Here we evaluate the origin of rebound virus in 16 ART-suppressed, chronically SIV-infected rhesus monkeys following ART discontinuation. We sequence viral RNA and viral DNA in these animals prior to ART initiation, during ART suppression, and following viral rebound, and we compare rebound viral RNA after ART discontinuation with near full-length viral DNA from peripheral blood and lymph node mononuclear cells (PBMC and LNMC) during ART suppression. Sequences of initial rebound viruses closely match viral DNA sequences in PBMC and LNMC during ART suppression. Recombinant viruses are rare in the initial rebound virus populations but arise quickly within 2–4 weeks after viral rebound. These data suggest that intact proviral DNA in PBMC and LNMC during ART suppression is likely the direct origin of viral rebound in chronically SIV-infected rhesus monkeys following ART discontinuation.

We sought to enhance the infectivity of three SHIV stocks by optimization of a key residue in human immunodeficiency virus type 1 (HIV-1) Env (Env375). We developed the following three new simian-human immunodeficiency virus (SHIV) stocks: SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH. SHIV-SF162p3S could not be optimized, SHIV-AE16W proved comparable to the parental virus, and SHIV-325cH demonstrated markedly enhanced replicative capacity compared with the parental virus.

The causal association of Zika virus (ZIKV) with microcephaly, congenital malformations in infants, and Guillain-Barré syndrome in adults highlights the need for effective vaccines. Thus far, efforts to develop ZIKV vaccines have focused on the viral envelope. ZIKV NS1 as a vaccine immunogen has not been fully explored, although it can circumvent the risk of antibody-dependent enhancement of ZIKV infection, associated with envelope antibodies. Here, we describe a novel DNA vaccine encoding a secreted ZIKV NS1, that confers rapid protection from systemic ZIKV infection in immunocompetent mice. We identify novel NS1 T cell epitopes in vivo and show that functional NS1-specific T cell responses are critical for protection against ZIKV infection. We demonstrate that vaccine-induced anti-NS1 antibodies fail to confer protection in the absence of a functional T cell response. This highlights the importance of using NS1 as a target for T cell-based ZIKV vaccines.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has spread to more than 70 countries worldwide since 2015. Despite active research, there are currently no licensed vaccines or therapeutics. We have previously reported the development of various adenoviral vectored vaccine candidates (ChAdOx1 ZIKV) with the ability to stimulate effective immunity in mice and provide protection upon a ZIKV challenge model, using a non-adjuvanted single vaccination approach. In this study, we constructed various modified vaccinia Ankara (MVA) viruses to express the ZIKV Envelope (E) with modifications on the precursor membrane (prM) or on the C-terminus envelope transmembrane domain (TM), similar to our ChAdOx1 vaccine candidates. MVA-ZIKV vaccine candidates were evaluated as a non-adjuvanted single vaccination regimen against a ZIKV Brazilian isolate, using viraemia as the correlate of protection. Here, we report the induction of a modest level of anti-ZIKV E antibodies by all MVA vectored vaccines and sub-optimal efficacy in a ZIKV challenge model. Our results indicate the requirement of additional strategies when using MVA-ZIKV vaccines to afford sterile protection upon a non-adjuvanted and single vaccination regime.

Adenoviral vectors have shown significant promise as vaccine delivery vectors due to their ability to elicit both innate and adaptive immune responses. α-defensins are effector molecules of the innate immune response and have been shown to modulate natural infection with adenoviruses, but the majority of α-defensin-adenovirus interactions studied to date have only been analyzed in vitro. In this study, we evaluated the role of α-defensin 5 (HD5) in modulating adenovirus vaccine immunogenicity using various serotype adenovirus vectors in mice. We screened a panel of human adenoviruses including Ad5 (species C), Ad26 (species D), Ad35 (species B), Ad48 (species D) and a chimeric Ad5HVR48 for HD5 sensitivity. HD5 inhibited transgene expression from Ad5 and Ad35 but augmented transgene expression from Ad26, Ad48, and Ad5HVR48. HD5 similarly suppressed antigen-specific IgG and CD8+ T cell responses elicited by Ad5 vectors in mice, but augmented IgG and CD8+ T cell responses and innate cytokine responses elicited by Ad26 vectors in mice. Moreover, HD5 suppressed the protective efficacy of Ad5 vectors but enhanced the protective efficacy of Ad26 vectors expressing SIINFEKL against a surrogate Listeria-OVA challenge in mice. These data demonstrate that HD5 differentially modulates adenovirus vaccine delivery vectors in a species-specific manner in vivo.

Maternal infection with Zika virus (ZIKV) can lead to microcephaly and other congenital abnormalities of the fetus. Although ZIKV vaccines that prevent or reduce viremia in non-pregnant mice have been described, a maternal vaccine that provides complete fetal protection would be desirable. Here, we show that adenovirus (Ad) vector-based ZIKV vaccines induce potent neutralizing antibodies that confer robust maternal and fetal protection against ZIKV challenge in pregnant, highly susceptible IFN-αβR−/− mice. Moreover, passive transfer of maternal antibodies from vaccinated dams protected pups against post-natal ZIKV challenge. These data suggest that Ad-based ZIKV vaccines may be able to provide protection in pregnant females against fetal ZIKV transmission in utero as well as in infants against ZIKV infection after birth.