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Background: The only HIV vaccine trial (RV144) to show any efficacy used a canary poxvirus vector and recombinant protein for immunisation; leading to the further exploration of poxviruses as HIV vaccine vectors. Our group has shown that heterologous prime boost immunisations of mice and macaques with a novel capripoxvirus vector, lumpy skin disease virus (LSDV) and modified vaccinia Ankara (MVA) induced high magnitude, broad cellular immune responses. Here, we present data on further modifications to the vaccines, which were tested in different regimens, for immunogenicity in rabbits. Methods: DNA (D), MVA (M) and LSDV (L) vaccines expressing HIV-1 CAP256SU gp150 and subtype C mosaic Gag ([Gag.sup.M]) were constructed. The expression of Env and Gag by all three vaccine was characterised in vitro. Groups of five rabbits were inoculated with four different vaccine regimens: DDMMLL; DDMLML; DDLMLM and DDLLMM at 0, 4, 8, 12, 16 and 20 weeks. The titres of binding and neutralising antibodies in the rabbit sera were determined. Results: The expression and secretion of HIV-1 CAP256SU gp150 and [Gag.sup.M] by all three vaccine vectors was verified in vitro. In addition, the formation of Gag virus-like particles containing gp150 was confirmed using negative stain electron microscopy and western blotting following density gradient centrifugation. The quaternary structures of gp150 on the cell surface was assessed by immunostaining with human monoclonal binding antibodies (MAbs) to HIV-1 Env, and fluorescent microscopy or FACS analysis. Binding of MAbs, PG16, PGT145 and CAP256 VRC26_08, confirmed that all three vaccines expressed some native, trimeric Env. All four vaccination regimens elicited high titres of neutralising antibodies (NAbs) to Tier 1A pseudovirus MW965.26 two weeks after the final immunisation. However, 5/5 rabbits in the DDLMLM, 3/5 in the DDLLMM, 2/5 in the DDMLML and none in the DDMMLL group developed NAbs to Tier 1B pseudovirus 6644. Two of the rabbits from the DDLMLM group also developed low levels of autologous Tier 2 NAbs. Conclusions: Low levels of autologous Tier 2 NAbs were induced in 2/5 rabbits inoculated with the DDLMLM vaccination regimen. Further studies are planned to determine if these vaccines elicit good cellular immune responses., OA15.01 R. Chapman (1); M. van Diepen (1); N. Douglass (1); S. Galant (1); E. Margolin (1); P. Ximba (1); T. Hermanus (2); P. Moore (2); E. Rybicki (3) and [...]

Background: SAAVI MVA-C was previously developed in South Africa and tested in Phase 1 clinical trials. Following more recent advances in antigen design, modifications were made to the HIV-1 genes expressed by modified vaccinia Ankara (MVA) and a head-to-head comparison was performed using the SAAVI MVA-C and newly constructed MVAGD5 vaccines. Methods: MVAGD5 was constructed with an HIV-1 subtype C mosaic gag inserted in the A11R-A12L locus and a modified form of the Du151 env gene inserted between I8R and G1L of MVA, both under vaccinia virus mH5 promoter control. The env gene was modified as follows: replacement of the native leader sequence with that of the human tissue plasminogen activator, replacement of the furin cleavage site with a flexible linker (2x GGGGS), inclusion of an 1559P mutation and truncation of gp160 to gp150. The two vaccines were compared for HIV-1 gene expression in HEK293T cells, by western blot analysis and immunofluorescence. Immunogenicity was tested in rabbits inoculated intramuscularly with 108pfu recombinant MVA at weeks 0 and 4; and 40 [micro]g soluble trimeric Du151 protein at week 12. Rabbits were bled at weeks 0, 4, 8, 12, 14 and 16 and serum was tested for binding antibodies by ELISA and neutralising antibodies using the TZM-bl pseudovirus assay. Results: Compared to SAAVI MVA-C the modified MVA vaccine, MVAGD5, showed increased expression of both Gag and Env in HEK293T cells. These two proteins were detected in the clarified medium from MVAGD5-infected cells, but not SAAVI MVA-C-infected cells. Co-localisation of Gag/Grttn and Env was observed by fluorescence microscopy in infected HEK293T cells for both MVAGD5/SAAVI MVA-C vaccines. MVAGD5 elicited binding antibodies after the first inoculation, whereas SAAVI MVA-C only elicited binding antibodies after the protein boost. MVAGD5 elicited neutralising antibodies to Tier 1A and Tier 1B pseudovirions whereas SAAVI MVA-C generated no significant neutralising response. Neither vaccine elicited autologous Tier 2 neutralising antibodies. Conclusions: Modifications made to both gag and env genes in the construction of SHIP MVAGD5, compared to the historic SAAVI MVA-C vaccine, resulted in superior in vitro expression of both HIV-1 genes and improved immunogenicity in rabbits., OA12.05 N. Douglass (1); M. Van Diepen (2); R. Chapman (2); S. Galant (2); E. Margolin (2); P. Ximba (2); T. Hermanus (3); P. Moore (3) and A.-L. Williamson (2) [...]

The book of Ruth is generally regarded as centring on the key figure of Ruth. It is claimed that this limits the narrative, influencing the interpretation. Several literary techniques suggest the author’s intentional focus on Naomi, her faith struggles and God’s gracious healing of an embittered mother. Whereas the paradigm of covenant faith and ḥesed [faithful love] feature prominently they do also highlight the spiritual transformation that came about for Naomi upon her return from Moab. The fullness and completed rest for Naomi continued consequentially to bear fruit for God’s people through the tribe of Judah. Historically, it confirmed the royal line of David which concluded with God’s Go’el [kinsman-redeemer].

We report the safety and immunogenicity of fractional and full dose Ad26.COV2.S and BNT162b2 in an open label phase 2 trial of participants previously vaccinated with a single dose of Ad26.COV2.S, with 91.4% showing evidence of previous SARS-CoV-2 infection. A total of 286 adults (with or without HIV) were enrolled >4 months after an Ad26.COV2.S prime and randomized 1:1:1:1 to receive either a full or half-dose booster of Ad26.COV2.S or BNT162b2 vaccine. B cell responses (binding, neutralization and antibody dependent cellular cytotoxicity-ADCC), and spike-specific T-cell responses were evaluated at baseline, 2, 12 and 24 weeks post-boost. Antibody and T-cell immunity targeting the Ad26 vector was also evaluated. No vaccine-associated serious adverse events were recorded. The full- and half-dose BNT162b2 boosted anti-SARS-CoV-2 binding antibody levels (3.9- and 4.5-fold, respectively) and neutralizing antibody levels (4.4- and 10-fold). Binding and neutralizing antibodies following half-dose Ad26.COV2.S were not significantly boosted. Full-dose Ad26.COV2.S did not boost binding antibodies but slightly enhanced neutralizing antibodies (2.1-fold). ADCC was marginally increased only after a full-dose BNT162b2. T-cell responses followed a similar pattern to neutralizing antibodies. Six months post-boost, antibody and T-cell responses had waned to baseline levels. While we detected strong anti-vector immunity, there was no correlation between anti-vector immunity in Ad26.COV2.S recipients and spike-specific neutralizing antibody or T-cell responses post-Ad26.COV2.S boosting. Overall, in the context of hybrid immunity, boosting with heterologous full- or half-dose BNT162b2 mRNA vaccine demonstrated superior immunogenicity 2 weeks post-vaccination compared to homologous Ad26.COV2.S, though rapid waning occurred by 12 weeks post-boost. Trial Registration: The study has been registered to the South African National Clinical Trial Registry (SANCTR): DOH-27-012022-7841. The approval letter from SANCTR has been provided in the up-loaded documents., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.Se. is a consultant for AstraZeneca Pharmaceuticals, Calyptus Pharmaceuticals, Inc, Darwin Health, EmerVax, EUROIMMUN, F. Hoffman-La Roche Ltd, Fortress Biotech, Gilead Sciences, Granite bio., Gritstone Oncology, Guggenheim Securities, Moderna, Pfizer, RiverVest Venture Partners, and Turnstone Biologics. A.G. is a consultant for Pfizer. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work. All other authors declare no competing interests., (Copyright: © 2024 Riou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The impact of previous SARS-CoV-2 infection on the durability of Ad26.COV2.S vaccine-elicited responses, and the effect of homologous boosting has not been well explored. We followed a cohort of healthcare workers for 6 months after receiving the Ad26.COV2.S vaccine and a further one month after they received an Ad26.COV2.S booster dose. We assessed longitudinal spike-specific antibody and T cell responses in individuals who had never had SARS-CoV-2 infection, compared to those who were infected with either the D614G or Beta variants prior to vaccination. Antibody and T cell responses elicited by the primary dose were durable against several variants of concern over the 6 month follow-up period, regardless of infection history. However, at 6 months after first vaccination, antibody binding, neutralization and ADCC were as much as 59-fold higher in individuals with hybrid immunity compared to those with no prior infection. Antibody cross-reactivity profiles of the previously infected groups were similar at 6 months, unlike at earlier time points, suggesting that the effect of immune imprinting diminishes by 6 months. Importantly, an Ad26.COV2.S booster dose increased the magnitude of the antibody response in individuals with no prior infection to similar levels as those with previous infection. The magnitude of spike T cell responses and proportion of T cell responders remained stable after homologous boosting, concomitant with a significant increase in long-lived early differentiated CD4 memory T cells. Thus, these data highlight that multiple antigen exposures, whether through infection and vaccination or vaccination alone, result in similar boosts after Ad26.COV2.S vaccination., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Moyo-Gwete et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neutralizing antibodies strongly correlate with protection for COVID-19 vaccines, but the corresponding memory B cells that form to protect against future infection are relatively understudied. Here we examine the effect of prior SARS-CoV-2 infection on the magnitude and phenotype of the memory B cell response to single dose Johnson and Johnson (Ad26.COV2.S) vaccination in South African health care workers. Participants were either naïve to SARS-CoV-2 or had been infected before vaccination. SARS-CoV-2-specific memory B-cells expand in response to Ad26.COV2.S and are maintained for the study duration (84 days) in all individuals. However, prior infection is associated with a greater frequency of these cells, a significant reduction in expression of the germinal center chemokine receptor CXCR5, and increased class switching. These B cell features correlated with neutralization and antibody-dependent cytotoxicity (ADCC) activity, and with the frequency of SARS-CoV-2 specific circulating T follicular helper cells (cTfh). Vaccination-induced effective neutralization of the D614G variant in both infected and naïve participants but boosted neutralizing antibodies against the Beta and Omicron variants only in participants with prior infection. In addition, the SARS-CoV-2 specific CD8+ T cell response correlated with increased memory B cell expression of the lung-homing receptor CXCR3, which was sustained in the previously infected group. Finally, although vaccination achieved equivalent B cell activation regardless of infection history, it was negatively impacted by age. These data show that phenotyping the response to vaccination can provide insight into the impact of prior infection on memory B cell homing, CSM, cTfh, and neutralization activity. These data can provide early signals to inform studies of vaccine boosting, durability, and co-morbidities., (© 2023. Springer Nature Limited.)

The kinetics of Fc-mediated functions following SARS-CoV-2 infection or vaccination in people living with HIV (PLWH) are not known. We compared SARS-CoV-2 spike-specific Fc functions, binding, and neutralization in PLWH and people without HIV (PWOH) during acute infection (without prior vaccination) with either the D614G or Beta variants of SARS-CoV-2, or vaccination with ChAdOx1 nCoV-19. Antiretroviral treatment (ART)-naïve PLWH had significantly lower levels of IgG binding, neutralization, and antibody-dependent cellular phagocytosis (ADCP) compared with PLWH on ART. The magnitude of antibody-dependent cellular cytotoxicity (ADCC), complement deposition (ADCD), and cellular trogocytosis (ADCT) was differentially triggered by D614G and Beta. The kinetics of spike IgG-binding antibodies, ADCC, and ADCD were similar, irrespective of the infecting variant between PWOH and PLWH overall. However, compared with PWOH, PLWH infected with D614G had delayed neutralization and ADCP. Furthermore, Beta infection resulted in delayed ADCT, regardless of HIV status. Despite these delays, we observed improved coordination between binding and neutralizing responses and Fc functions in PLWH. In contrast to D614G infection, binding responses in PLWH following ChAdOx-1 nCoV-19 vaccination were delayed, while neutralization and ADCP had similar timing of onset, but lower magnitude, and ADCC was significantly higher than in PWOH. Overall, despite delayed and differential kinetics, PLWH on ART develop comparable responses to PWOH, supporting the prioritization of ART rollout and SARS-CoV-2 vaccination in PLWH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Motsoeneng, Manamela, Kaldine, Kgagudi, Hermanus, Ayres, Makhado, Moyo-Gwete, van der Mescht, Abdullah, Boswell, Ueckermann, Rossouw, Madhi, Moore and Richardson.)

Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity., Competing Interests: Declaration of interests K.A.P. and I.S.G. are listed as inventors on patents filed describing the antibodies discovered here. A.R.S. and I.S.G. are co-founders of AbSeek Bio. J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines, and is founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics, and AstraZeneca. The Georgiev laboratory at Vanderbilt University Medical Center has received unrelated funding from Takeda Pharmaceuticals., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The combination of mosaic Gag and CAP256 envelope in an HIV vaccine regimen comprising DNA prime and modified vaccinia Ankara (MVA) boost followed by protein boost has previously been shown to generate robust autologous Tier 2 neutralizing antibodies (nAbs) in rabbits. Further refinements of this strategy have been investigated to improve antibody responses. The delivery of both DNA and recombinant MVA vaccines with a needle-free device was compared to delivery by injection, and the effect of formulating the DNA vaccine with adjuvant CpG ODN 1826 was determined. The Pharmajet Stratis ® needle-free injection device (PharmaJet, Golden, CO, USA) improved binding antibody responses to the DNA vaccine as well as both binding and neutralizing antibody responses to the MVA vaccines. Formulation of the DNA vaccines with CpG adjuvant further improved the antibody responses. A shortened vaccination regimen of a single DNA inoculation followed by a single MVA inoculation did not elicit Tier 1B nor Tier 2 neutralization responses as produced by the two DNA, followed by two MVA vaccination regimen. This study showed the immunogenicity of HIV DNA and MVA vaccines administered in a DDMM regimen could be improved using the PharmaJet Stratis needle-free injection device and formulation of the DNA vaccines with CpG adjuvant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.4 and BA.5 variants caused major waves of infections. Here, we assess the sensitivity of BA.4 to binding, neutralization, and antibody-dependent cellular cytotoxicity (ADCC) potential, measured by FcγRIIIa signaling, in convalescent donors infected with four previous variants of SARS-CoV-2, as well as in post-vaccination breakthrough infections (BTIs) caused by Delta or BA.1. We confirm that BA.4 shows high-level neutralization resistance regardless of the infecting variant. However, BTIs retain activity against BA.4, albeit at reduced titers. BA.4 sensitivity to ADCC is reduced compared with other variants but with smaller fold losses compared with neutralization and similar patterns of cross-reactivity. Overall, the high neutralization resistance of BA.4, even to antibodies from BA.1 infection, provides an immunological mechanism for the rapid spread of BA.4 immediately after a BA.1-dominated wave. Furthermore, although ADCC potential against BA.4 is reduced, residual activity may contribute to observed protection from severe disease., Competing Interests: Declaration of interests P.L.M. is a member of the advisory board for Cell Reports Medicine., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Antibody Mediated Prevention trials showed that the broadly neutralizing antibody (bnAb) VRC01 prevented acquisition of human immunodeficiency virus-1 (HIV-1) sensitive to VRC01. Using AMP trial data, here we show that the predicted serum neutralization 80% inhibitory dilution titer (PT 80 ) biomarker-which quantifies the neutralization potency of antibodies in an individual's serum against an HIV-1 isolate-can be used to predict HIV-1 prevention efficacy. Similar to the results of nonhuman primate studies, an average PT 80 of 200 (meaning a bnAb concentration 200-fold higher than that required to reduce infection by 80% in vitro) against a population of probable exposing viruses was estimated to be required for 90% prevention efficacy against acquisition of these viruses. Based on this result, we suggest that the goal of sustained PT 80 <200 against 90% of circulating viruses can be achieved by promising bnAb regimens engineered for long half-lives. We propose the PT 80 biomarker as a surrogate endpoint for evaluatinon of bnAb regimens, and as a tool for benchmarking candidate bnAb-inducing vaccines., (© 2022. The Author(s).)

Induction of broadly neutralizing antibodies (bNAbs) to HIV and other diverse pathogens will likely require the use of multiple immunogens. An understanding of the dynamics of antibody development to multiple diverse but related antigens would facilitate the rational design of immunization strategies. Here, we characterize, in detail, the development of neutralizing antibodies in three individuals coinfected with several divergent HIV variants. Two of these coinfected individuals developed additive or cross-neutralizing antibody responses. However, interference was observed in the third case, with neutralizing antibody responses to one viral variant arising to the near exclusion of neutralizing responses to the other. Longitudinal characterization of the diversity in the Envelope glycoprotein trimer (Env) structure showed that in the individual who developed the broadest neutralizing antibodies, circulating viruses shared a conserved epitope on the trimer apex that was targeted by cross-neutralizing antibodies. In contrast, in the other two individuals, diversity was distributed across Env. Taken together, these data highlight that multiple related immunogens can result in immune interference. However, they also suggest that immunogen cocktails presenting shared, conserved neutralizing epitopes in a variable background may focus broadly neutralizing antibody responses to these targets. IMPORTANCE Despite being the focus of extensive research, we still do not know how to reproducibly elicit cross-neutralizing antibodies against variable pathogens by vaccination. Here, we characterize the antibody responses in people coinfected with more than one HIV variant, providing insights into how the use of antigen "cocktails" might affect the breadth of the elicited neutralizing antibody response and how the relatedness of the antigens may shape this.

The Johnson and Johnson Ad26.COV2.S single-dose vaccine represents an attractive option for coronavirus disease 2019 (COVID-19) vaccination in countries with limited resources. We examined the effect of prior infection with different SARS-CoV-2 variants on Ad26.COV2.S immunogenicity. We compared participants who were SARS-CoV-2 naive with those either infected with the ancestral D614G virus or infected in the second wave when Beta predominated. Prior infection significantly boosts spike-binding antibodies, antibody-dependent cellular cytotoxicity, and neutralizing antibodies against D614G, Beta, and Delta; however, neutralization cross-reactivity varied by wave. Robust CD4 and CD8 T cell responses are induced after vaccination, regardless of prior infection. T cell recognition of variants is largely preserved, apart from some reduction in CD8 recognition of Delta. Thus, Ad26.COV2.S vaccination after infection could result in enhanced protection against COVID-19. The impact of the infecting variant on neutralization breadth after vaccination has implications for the design of second-generation vaccines based on variants of concern., Competing Interests: Declaration of interests A.S. is a consultant for Gritstone, Flow Pharma, CellCarta, Arcturus, Oxford Immunotech, and Avalia. All of the other authors declare no competing interests. LJI has filed for patent protection for various aspects of vaccine design and identification of specific epitopes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The modest protective effects of the RV144 HIV-1 vaccine trial have prompted the further exploration of improved poxvirus vector systems that can yield better immune responses and protection. In this study, a recombinant lumpy skin disease virus (LSDV) expressing HIV-1 CAP256.SU gp150 (Env) and a subtype C mosaic Gag was constructed (LSDVGC5) and compared to the equivalent recombinant modified vaccinia Ankara (MVAGC5). In vitro characterization confirmed that cells infected with recombinant LSDV produced Gag virus-like particles containing Env, and that Env expressed on the surface of the cells infected with LSDV was in a native-like conformation. This candidate HIV-1 vaccine (L) was tested in a rabbit model using different heterologous vaccination regimens, in combination with DNA (D) and MVA (M) vectors expressing the equivalent HIV-1 antigens. The four different vaccination regimens (DDMMLL, DDMLML, DDLMLM, and DDLLMM) all elicited high titers of binding and Tier 1A neutralizing antibodies (NAbs), and some regimens induced Tier 1B NAbs. Furthermore, two rabbits in the DDLMLM group developed low levels of autologous Tier 2 NAbs. The humoral immune responses elicited against HIV-1 Env by the recombinant LSDVGC5 were comparable to those induced by MVAGC5.

Objectives: Antibody function has been extensively studied in HIV-infected adults but is relatively understudied in children. Emerging data suggests enhanced development of broadly neutralizing antibodies (bNAbs) in children but Fc effector functions in this group are less well defined. Here, we profiled overall antibody function in HIV-infected children., Design: Plasma samples from a cross-sectional study of 50 antiretroviral therapy-naive children (aged 1-11 years) vertically infected with HIV-1 clade A were screened for HIV-specific binding antibody levels and neutralizing and Fc-mediated functions., Methods: Neutralization breadth was determined against a globally representative panel of 12 viruses. HIV-specific antibody levels were determined using a multiplex assay. Fc-mediated antibody functions measured were antibody-dependent: cellular phagocytosis (ADCP); neutrophil phagocytosis (ADNP); complement deposition (ADCD) and natural killer function (ADNK)., Results: All children had HIV gp120-specific antibodies, largely of the IgG1 subtype. Fifty-four percent of the children exhibited more than 50% neutralization breadth, with older children showing significantly broader neutralization activity. Apart from ADCC, observed only in 16% children, other Fc-mediated functions were common (>58% children). Neutralization breadth correlated with Fc-mediated functions suggesting shared determinants of enhanced antibody function exist., Conclusions: These results are consistent with previous observations that children may develop high levels of neutralization breadth. Furthermore, the striking association between neutralization breadth and Fc effector function suggests that HIV vaccination in children could yield multifunctional antibodies. Paediatric populations may therefore provide an ideal window of opportunity for HIV vaccination strategies., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

A leading strategy for developing a prophylactic HIV-1 vaccine is the elicitation of antibodies that can neutralize a large fraction of circulating HIV-1 variants. However, a major challenge that has limited the effectiveness of current vaccine candidates is the extensive global diversity of the HIV-1 envelope protein (Env), the sole target for HIV-neutralizing antibodies. To address this challenge, various strategies incorporating Env diversity into the vaccine formulation have been proposed. Here, we assessed the potential of two such strategies that utilize a nanoparticle-based vaccine platform to elicit broadly neutralizing antibody responses. The nanoparticle immunogens developed here consisted of different formulations of Envs from strains BG505 (clade A) and CZA97 (clade C), attached to the N-termini of bacterial ferritin. Single-antigen nanoparticle cocktails, as well as mosaic nanoparticles bearing both Env trimers, elicited high antibody titers in mice and guinea pigs. Furthermore, serum from guinea pigs immunized with nanoparticle immunogens achieved autologous, and in some cases heterologous, tier 2 neutralization, although significant differences between mosaic and single-antigen nanoparticles were not observed. These results provide insights into the ability of different vaccine strategies for incorporating Env sequence diversity to elicit neutralizing antibodies, with implications for the development of broadly protective HIV-1 vaccines.

SARS-CoV-2 501Y.V2 (B.1.351), a novel lineage of coronavirus causing COVID-19, contains substitutions in two immunodominant domains of the spike protein. Here, we show that pseudovirus expressing 501Y.V2 spike protein completely escapes three classes of therapeutically relevant antibodies. This pseudovirus also exhibits substantial to complete escape from neutralization, but not binding, by convalescent plasma. These data highlight the prospect of reinfection with antigenically distinct variants and foreshadows reduced efficacy of spike-based vaccines.

Broadly neutralizing antibodies (bNAbs) may constitute an essential component of a protective vaccine against HIV-1, yet no immunogen has been able to elicit them. To characterize the development of bNAbs in HIV-1 subtype C infected individuals, a panel of 18 Env-pseudotyped viruses was used to screen 18 study participants. The specificity of plasma neutralization was mapped against Env mutants and MPER chimeras. Envelope (env) gene sequence evolution was characterized by single genome amplification and sequencing. Three out of eighteen individuals developed broad plasma neutralizing activity (>60% breadth). Two of the three participants may target epitopes comprising glycans at position 276 of the D loop in the CD4 binding site and 332 glycan supersite, respectively. Deletion of these glycans was associated with neutralization resistance. Our study describes the kinetics of the development of plasma neutralizing activity and identified amino acid residue changes suggestive of immune pressure on putative epitopes. The study enhances our understanding of how neutralization breadth develops in the course of HIV-1 subtype C infection., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The HIV-1 envelope glycoprotein (Env) is present on the surface of the virion at a very low density compared to most other enveloped viruses. Substitution of various parts of the stalk domain of Env (gp41) with the corresponding elements from other viral glycoproteins has been shown to increase Env spike density on the cell membrane and surface of virus-like particles (VLPs). In this study, chimeric Env antigens were generated by replacing the transmembrane and cytoplasmic domains of HIV-1 Env with the corresponding regions from the influenza H5 hemagglutinin (HA) (gp140HA 2 tr) and by replacing the entire gp41 region of Env with the HA 2 subunit of HA (gp120HA 2 ). Recombinant DNA and modified vaccinia Ankara (MVA) vaccines expressing HIV-1 subtype C mosaic Gag and gp150 Env or either of the chimeras were generated. Surprisingly, no significant differences were found in the levels of expression of gp150 Env or either of the chimeras on the surface of cells or on Gag VLPs. Differences were, however, observed in the binding of different monoclonal antibodies to the HIV-1 Env. Monoclonal antibodies, which recognized a V1 / V2 quaternary epitope at the tip of the native Env trimer, bound gp150 and gp140HA 2 tr chimera but failed to bind to the gp120HA 2 chimera. Autologous Tier 2 neutralizing antibodies (NAbs) were produced by rabbits inoculated with DNA and MVA vaccines expressing the gp140HA 2 tr chimera or gp150 Env, but not those immunized with the gp120HA 2 Env. These results showed that the addition of an HA 2 stalk to HIV-1 gp120 did not improve immunogenicity, but rather that the full-length gp150 was required for optimal presentation of epitopes for the elicitation of a neutralizing antibody response to HIV-1., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen., (Copyright © 2019 Margolin, Chapman, Meyers, van Diepen, Ximba, Hermanus, Crowther, Weber, Morris, Williamson and Rybicki.)

With the HIV-1 epidemic in southern Africa still rising, a prophylactic vaccine against the region's most prolific subtype (subtype C) would be a significant step forward. In this paper we report on the effect of 2 different adjuvants, AddaVax and AlhydroGel, formulated with HIV-1 subtype C gp140, on the development of binding and neutralising antibody titres in rabbits. AddaVax is a squalene-based oil-in-water nano-emulsion (similar to MF59) which can enhance both cellular and humoral immune responses, whilst AlhydroGel (aluminium hydroxide gel) mainly drives a Th2 response. The gp140 gene tested was derived from the superinfecting virus (SU) from participant CAP256 in the CAPRISA 002 Acute infection cohort. The furin cleavage site of the Env protein was replaced with a flexible linker and an I559P mutation introduced. Lectin affinity purified soluble Env protein was mainly trimeric as judged by molecular weight using BN-PAGE and contained intact broadly neutralising epitopes for the V3-glycan supersite (monoclonal antibodies PGT128 and PGT135), the CD4 binding site (VRC01) and the V2-glycan (PG9) but not for the trimer-specific monoclonal antibodies PG16, PGT145 and CAP256-VRC26&#95;08. When this soluble Env protein was tested in rabbits, AlhydroGel significantly enhanced soluble Env and V1V2 binding antibodies when compared to AddaVax. Finally, AlhydroGel resulted in significantly higher neutralization titres for a subtype C Tier 1A virus (MW965.26) and increased neutralization breadth to Tier 1A and 1B viruses. However, no autologous Tier 2 neutralisation was observed. These data suggest that adjuvant selection is critical for developing a successful vaccine and AlhydroGel should be further investigated. Additional purification of trimeric native-like CAP256 Env and/or priming with DNA or MVA might enhance the induction of neutralizing antibodies and possible Tier 2 HIV-1 neutralisation., Competing Interests: The authors have declared that no competing interests exist.

Antibodies that bind residue K169 in the V2 region of the HIV-1 envelope correlated with reduced risk of infection in the RV144 vaccine trial but were restricted to two ED-motif-encoding light chain genes. Here, we identify an HIV-infected donor with high-titer V2 peptide-binding antibodies and isolate two antibody lineages (CAP228-16H/19F and CAP228-3D) that mediate potent antibody-dependent cell-mediated cytotoxicity (ADCC). Both lineages use the IGHV5-51 heavy chain germline gene, similar to the RV144 antibody CH58, but one lineage (CAP228-16H/19F) uses a light chain without the ED motif. A cocrystal structure of CAP228-16H bound to a V2 peptide identified a IGLV3-21 gene-encoded DDxD motif that is used to bind K169, with a mechanism that allows CAP228-16H to recognize more globally relevant V2 immunotypes. Overall, these data further our understanding of the development of cross-reactive, V2-binding, antiviral antibodies and effectively expand the human light chain repertoire able to respond to RV144-like immunogens., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The safety and immunogenicity of SAAVI DNA-C2 (4 mg IM), SAAVI MVA-C (2.9 x 109 pfu IM) and Novartis V2-deleted subtype C gp140 (100 mcg) with MF59 adjuvant in various vaccination regimens was evaluated in HIV-uninfected adults in South Africa., Methods: Participants at three South African sites were randomized (1:1:1:1) to one of four vaccine regimens: MVA prime, sequential gp140 protein boost (M/M/P/P); concurrent MVA/gp140 (MP/MP); DNA prime, sequential MVA boost (D/D/M/M); DNA prime, concurrent MVA/gp140 boost (D/D/MP/MP) or placebo. Peak HIV specific humoral and cellular responses were measured., Results: 184 participants were enrolled: 52% were female, all were Black/African, median age was 23 years (range, 18-42 years) and 79% completed all vaccinations. 159 participants reported at least one adverse event, 92.5% were mild or moderate. Five, unrelated, serious adverse events were reported. The M/M/P/P and D/D/MP/MP regimens induced the strongest peak neutralizing and binding antibody responses and the greatest CD4+ T-cell responses to Env. All peak neutralizing and binding antibody responses decayed with time. The MVA, but not DNA, prime contributed to the humoral and cellular immune responses. The D/D/M/M regimen was poorly immunogenic overall but did induce modest CD4+ T-cell responses to Gag and Pol. CD8+ T-cell responses to any antigen were low for all regimens., Conclusions: The SAAVI DNA-C2, SAAVI MVA-C and Novartis gp140 with MF59 adjuvant in various combinations were safe and induced neutralizing and binding antibodies and cellular immune responses. Sequential immunization with gp140 boosted immune responses primed by MVA or DNA. The best overall immune responses were seen with the M/M/P/P regimen., Trial Registration: ClinicalTrials.gov NCT01418235., Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: MA and MP are employed by the National Institute of Allergy and Infectious Diseases (NIAID), the study sponsor. GC, KM, SK, CW, A-LW, LM, GDT, SCD, PG, NG, CY, GG, LGB, DCM, JK, LC were recipients of NIAID funding, and this publication is a result of activities funded by NIAID. MA and MP were not involved with the process of funding these awards, nor in their administration or scientific aspects, and, in accordance with NIAID policies, are deferred from decisions regarding funding of coauthors for a requisite period. This study was also partly funded by Novartis Vaccines. At the time the study was conducted, Susan W. Barnett was an employee and stakeholder of Novartis Vaccines and Diagnostics. This does not alter our adherence to PLOS ONE policies on sharing data and materials (as detailed online in the guide for authors, http://www.PLOSone.org/static/editorial.action#competing).

The glycan supersite centered on N332 in the V3 base of the HIV-1 envelope (Env) is a target for broadly neutralizing antibodies (bnAbs) such as PGT121 and PGT128. In this study, we examined the basis of resistance of HIV-1 clade C Envs obtained from broadly cross neutralizing (BCN) plasma of an Indian donor with N332 specificity. Pseudotyped viruses expressing autologous envs were found to be resistant to autologous BCN plasma as well as to PGT121 and PGT128 mAbs despite the majority of Envs containing an intact N332 residue. While resistance of one of the Envs to neutralization by autologous plasma antibodies with shorter V1 loop length was found to be correlated with a N332S mutation, resistance to neutralization of rest of the Envs was found to be associated with longer V1 loop length and acquisition of protective N-glycans. In summary, we show evidence of escape of circulating HIV-1 clade C in an individual from autologous BCN antibodies by three distinct mechanisms.

Broadly cross-neutralizing (BCN) antibodies are likely to be critical for an effective HIV vaccine. However, the ontogeny of such antibodies and their relationship with autologous viral evolution is unclear. Here, we characterized viral evolution in CAP256, a subtype C-infected individual who developed potent BCN antibodies targeting positions R166 and K169 in the V2 region. CAP256 was superinfected at 3 months postinfection with a virus that was highly sensitive to BCN V2-dependent monoclonal antibodies. The autologous neutralizing response in CAP256 was directed at V1V2, reaching extremely high titers (>1:40,000) against the superinfecting virus at 42 weeks, just 11 weeks prior to the development of the BCN response targeting the same region. Recombination between the primary and superinfecting viruses, especially in V2 and gp41, resulted in two distinct lineages by 4 years postinfection. Although neutralization of some CAP256 clones by plasma from as much as 2 years earlier suggested incomplete viral escape, nonetheless titers against later clones were reduced at least 40-fold to less than 1:1,000. Escape mutations were identified in each lineage, either at R166 or at K169, suggesting that strain-specific and BCN antibodies targeted overlapping epitopes. Furthermore, the early dependence of CAP256 neutralizing antibodies on the N160 glycan decreased with the onset of neutralization breadth, indicating a change in specificity. These data suggest rapid maturation, within 11 weeks, of CAP256 strain-specific antibodies to acquire breadth, with implications for the vaccine elicitation of BCN V2-dependent antibodies. Overall these studies demonstrate that ongoing viral escape is possible, even from BCN antibodies.

Neutralizing (nAbs) and high affinity binding antibodies may be critical for an efficacious HIV-1 vaccine. We characterized virus-specific nAbs and binding antibody responses over 21 months in eight HIV-1 subtype C chronically infected individuals with heterogeneous rates of disease progression. Autologous nAb titers of study exit plasma against study entry viruses were significantly higher than contemporaneous responses at study entry (p=0.002) and exit (p=0.01). NAb breadth and potencies against subtype C viruses were significantly higher than for subtype A (p=0.03 and p=0.01) or B viruses (p=0.03; p=0.05) respectively. Gp41-IgG binding affinity was higher than gp120-IgG (p=0.0002). IgG-FcγR1 affinity was significantly higher than FcγRIIIa (p<0.005) at study entry and FcγRIIb (p<0.05) or FcγRIIIa (p<0.005) at study exit. Evolving IgG binding suggests alteration of immune function mediated by binding antibodies. Evolution of nAbs was a potential marker of HIV-1 disease progression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful, a minority of individuals naturally develop these antibodies after many years of infection. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly under-represented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.

An understanding of how broadly neutralizing activity develops in HIV-1-infected individuals is needed to guide vaccine design and immunization strategies. Here we used a large panel of 44 HIV-1 envelope variants (subtypes A, B, and C) to evaluate the presence of broadly neutralizing antibodies in serum samples obtained 3 years after seroconversion from 40 women enrolled in the CAPRISA 002 acute infection cohort. Seven of 40 participants had serum antibodies that neutralized more than 40% of viruses tested and were considered to have neutralization breadth. Among the samples with breadth, CAP257 serum neutralized 82% (36/44 variants) of the panel, while CAP256 serum neutralized 77% (33/43 variants) of the panel. Analysis of longitudinal samples showed that breadth developed gradually starting from year 2, with the number of viruses neutralized as well as the antibody titer increasing over time. Interestingly, neutralization breadth peaked at 4 years postinfection, with no increase thereafter. The extent of cross-neutralizing activity correlated with CD4(+) T cell decline, viral load, and CD4(+) T cell count at 6 months postinfection but not at later time points, suggesting that early events set the stage for the development of breadth. However, in a multivariate analysis, CD4 decline was the major driver of this association, as viral load was not an independent predictor of breadth. Mapping of the epitopes targeted by cross-neutralizing antibodies revealed that in one individual these antibodies recognized the membrane-proximal external region (MPER), while in two other individuals, cross-neutralizing activity was adsorbed by monomeric gp120 and targeted epitopes that involved the N-linked glycan at position 332 in the C3 region. Serum antibodies from the other four participants targeted quaternary epitopes, at least 2 of which were PG9/16-like and depended on the N160 and/or L165 residue in the V2 region. These data indicate that fewer than 20% of HIV-1 subtype C-infected individuals develop antibodies with cross-neutralizing activity after 3 years of infection and that these antibodies target different regions of the HIV-1 envelope, including as yet uncharacterized epitopes.

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers. Repair of the FN/LRD-K-K motif in resistant heterologous viruses conferred sensitivity, with titers sometimes exceeding 1:10,000. Comparison of the CAP256 epitope with that of the PG9/PG16 monoclonal antibodies suggested that these epitopes overlapped, adding to the mounting evidence that this may represent a common neutralization target that should be further investigated as a potential vaccine candidate.