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The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design.

One of the strategies towards an effective HIV-1 vaccine is to elicit broadly neutralizing antibody responses that target the high HIV-1 Env diversity. Here, we present an HIV-1 vaccine candidate that consists of cobalt porphyrin-phospholipid (CoPoP) liposomes decorated with repaired and stabilized clade C HIV-1 Env trimers in a prefusion conformation. These particles exhibit high HIV-1 Env trimer decoration, serum stability and bind broadly neutralizing antibodies. Three sequential immunizations of female rabbits with CoPoP liposomes displaying a different clade C HIV-1 gp140 trimer at each dosing generate high HIV-1 Env-specific antibody responses. Additionally, serum neutralization is detectable against 18 of 20 multiclade tier 2 HIV-1 strains. Furthermore, the peak antibody titers induced by CoPoP liposomes can be recalled by subsequent heterologous immunization with Ad26-encoded membrane-bound stabilized Env antigens. Hence, a CoPoP liposome-based HIV-1 vaccine that can generate cross-clade neutralizing antibody immunity could potentially be a component of an efficacious HIV-1 vaccine. HIV vaccine candidates often have limited capacity to induce broadly neutralizing antibodies (nAbs). In this study, the authors show that stabilized Clade C HIV-1 Env protein trimers decorated on CoPoP liposomes induce nAbs against 18 of 20 multiclade tier 2 HIV-1 strains in immunized rabbits.

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1’s extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination.

Current SARS-CoV-2 vaccines are effective, but long-term protection is threatened by the emergence of virus variants. We generated a virosome vaccine containing the Beta spike protein and compared its immunogenicity in mice to a virosome vaccine containing the original Wuhan spike. Two administrations of the virosomes induced potent SARS-CoV-2 neutralizing antibodies in both vaccine groups. The level of autologous neutralization in Beta-vaccinated mice was similar to the level of autologous neutralization in Wuhan-vaccinated mice. However, heterologous neutralization to the Wuhan strain in Beta-vaccinated mice was 4.7-fold lower than autologous neutralization, whereas heterologous neutralization to the Beta strain in Wuhan-vaccinated mice was reduced by only 1.9-fold compared to autologous neutralization levels. In addition, neutralizing activity against the D614G, Alpha and Delta variants was also significantly lower after Beta spike vaccination than after Wuhan spike vaccination. Our results show that Beta spike vaccination induces inferior neutralization breadth. These results are informative for programs aimed to develop broadly active SARS-CoV-2 vaccines.

Gold nanoparticles (AuNPs) decorated with antigens have recently emerged as promising tools for vaccine development due to their innate ability to provide stability to antigens and modulate immune responses. In this study, we have engineered deactivated virus-like particles (VLPs) by precisely functionalizing gold cores with coronas comprising the full SARS-CoV-2 spike protein (S). Using BALB/c mice as a model, we investigated the immunogenicity of these S-AuNPs-VLPs. Our results demonstrate that S-AuNPs-VLPs consistently enhanced antigen-specific antibody responses compared to the S protein free in solution. This enhancement included higher binding antibody titers, higher neutralizing capacity of antibodies, and stronger T-cell responses. Compared to the mRNA COVID-19 vaccine, where the S protein is synthesized in situ, S-AuNPs-VLPs induced comparable binding and neutralizing antibody responses, but substantially superior T-cell responses. In conclusion, our study highlights the potential of conjugated AuNPs as an effective antigen-delivery system for protein-based vaccines targeting a broad spectrum of infectious diseases and other emergent viruses.

Broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) have the capacity to delay viral rebound when administered to people with HIV-1 (PWH) during anti-retroviral therapy (ART) interruption. To further enhance the performance of bNAbs through their Fc effector functions, in particular NK cell-mediated killing of HIV-1 infected cells, we have produced a panel of glyco-engineered (afucosylated) bNAbs with enhanced affinity for Fc gamma receptor IIIa. These afucosylated anti-HIV-1 bNAbs enhance NK cell activation and degranulation compared to fucosylated counterparts even at low antigen density. NK cells from PWH expressing exhaustion markers PD-1 and TIGIT are activated in a similar fashion by afucosylated bNAbs as NK cell from HIV-1 negative individuals. Killing of HIV-1 infected cells is most effective with afucosylated bNAbs 2G12, N6, PGT151 and PGDM1400, whereas afucosylated PGT121 and non-neutralizing antibody A32 only induce minor NK cell-mediated killing. These data indicate that the approach angle and affinity of Abs influence the capacity to induce antibody-dependent cellular cytotoxicity. Thus, afucosylated bNAbs have the capacity to induce NK cell-mediated killing of infected cells, which warrants further investigation of afucosylated bNAb administration in vivo, aiming for reduction of the viral reservoir and ART free durable control. A study on glyco-engineered anti-HIV-1 antibodies suggests that afucosylated bNAbs have the capacity to induce killing of HIV-1 infected cells at low antigen density despite NK cell exhaustion.

The HIV-1 envelope glycoprotein trimer is poorly immunogenic because it is covered by a dense glycan shield. As a result, recombinant Env glycoproteins generally elicit inadequate antibody levels that neutralize clinically relevant, neutralization-resistant (Tier-2) HIV-1 strains. Multivalent antigen presentation on nanoparticles is an established strategy to increase vaccine-driven immune responses. However, due to nanoparticle instability in vivo, the display of non-native Env structures, and the inaccessibility of many neutralizing antibody (NAb) epitopes, the effects of nanoparticle display are generally modest for Env trimers. Here, we generate two-component self-assembling protein nanoparticles presenting twenty SOSIP trimers of the clade C Tier-2 genotype 16055. We show in a rabbit immunization study that these nanoparticles induce 60-fold higher autologous Tier-2 NAb titers than the corresponding SOSIP trimers. Epitope mapping studies reveal that the presentation of 16055 SOSIP trimers on these nanoparticle focuses antibody responses to an immunodominant apical epitope. Thus, these nanoparticles are a promising platform to improve the immunogenicity of Env trimers with apex-proximate NAb epitopes.

The SARS-CoV-2 pandemic revealed the rapid evolution of circulating strains. This led to new variants carrying mostly mutations within the receptor binding domain, which is immunodominant upon immunization and infection. In order to steer the immune response away from RBD epitopes to more conserved domains, we generated S glycoprotein trimers without RBD and stabilized them by formaldehyde cross-linking. The cryoEM structure demonstrated that SΔRBD folds into the native prefusion conformation, stabilized by one specific cross-link between S2 protomers. SΔRBD was coated onto lipid vesicles, to produce synthetic virus-like particles, SΔRBD-LV, which were utilized in a heterologous prime-boost strategy. Immunization of cynomolgus macaques either three times with the mRNA Comirnaty vaccine or two times followed by SΔRBD-LV showed that the SΔRBD-LV boost induced similar antibody titers and neutralization of different variants, including omicron. Upon challenge with omicron XBB.3, both the Comirnaty only and Comirnaty/SΔRBD-LV vaccination schemes conferred similar overall protection from infection for both the Comirnaty only and Comirnaty/SΔRBD-LV vaccination schemes. However, the SΔRBD-LV boost indicated better protection against lung infection than the Comirnaty strategy alone. Together our findings indicate that SΔRBD is highly immunogenic and provides improved protection compared to a third mRNA boost indicative of superior antibody-based protection.

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

The immunogenicity of HIV-1 envelope (Env) trimers is generally poor. We used the clinically relevant ConM SOSIP trimer to compare the ability of different adjuvants (squalene emulsion, ISCOMATRIX, GLA-LSQ, and MPLA liposomes) to support neutralizing antibody (NAb) responses in rabbits. The trimers were administered as free proteins or on nanoparticles. The rank order for the adjuvants was ISCOMATRIX > SE > GLA-LSQ ~ MPLA liposomes > no adjuvant. Stronger NAb responses were elicited when the ConM SOSIP trimers were presented on ferritin nanoparticles. We also found that the GLA-LSQ adjuvant induced an unexpectedly strong antibody response to the ferritin core of the nanoparticles. This “off-target” effect may have compromised its ability to induce the more desired antitrimer antibodies. In summary, both adjuvants and nanoparticle display can improve the magnitude of the antibody response to SOSIP trimers but the best combination of trimer presentation and adjuvant can only be identified experimentally.