Publications

Abstract Background We have previously isolated a highly mutated (83% homologous to predicted heavy chain germline) antibody (Ab) termed C group 76-Q13-6F5 (6F5) that targets a conformational epitope on gp41. 6F5, though non-neutralizing, has the capacity to mediate Ab dependent cell cytotoxicity (ADCC). When the variable chain (predicted to be VH1-02 derived) was mutated to germline (termed C group 76 ancestor, or 76Canc), surprisingly this Ab still exhibited significant ADCC activity. Many HIV vaccine strategies are focused on raising highly mutated Abs. We propose that there would be an advantage to developing vaccines related to epitopes that permit functional targeting by Abs using germline variable gene sequences. Methods To explore potential protein targets for vaccination strategies to raise and develop such Abs, we interrogated a peptide array of 29,127 linear peptides using PEPperCHIP® Human Epitome Microarray. We then confirmed peptide binding by Western blot and ELISAs. We also assessed binding to CDI laboratories HuProt protein microarray, containing > 21,000 human proteins. Results 76Canc specifically recognized a number of peptides enriched for glutamic and aspartic acid residues (top hit DEEEEYDEDEYEYDE). Meme analysis of positive peptides revealed a peptide sequence most similar to Hepatitis C virus, similar to a peptide implicated in Kawasaki disease (KD). We confirmed specific binding of four of the top peptide hits, including hepatitis C peptide recognition. We then confirmed binding of 76Canc-related Abs to a published optimized KD related peptide (KPAVIPDREALYQDIDEMEEC). Serum from KD and infectious controls was used to compete with biotinylated 76Canc-related Abs. Serum Abs targeting this epitope showed no specific correlation to having KD. Autoantigen screening of 76Canc identified a single human protein of interest that did contain acidic amino acid rich regions.Figure 1:HIV-1 gp41 antibodies recognize peptides similar to peptide implicated in Kawasaki Disease Conclusion This study reveals acidic motif targeting by specific anti-gp41 Abs and the derived germline Ab, but no evidence that these Abs are related to inflammation similar to KD. Cautious development of targeting such Abs by vaccination is warranted. Future structural comparison of these peptides with native proteins and binding competition studies are needed to confirm mimotope binding. Disclosures Mark D. Hicar, MD/PhD, Pfizer: site investigator for 2 trial

Abstract The threat of spillovers of coronaviruses associated with the severe acute respiratory syndrome (SARS) from animals to humans necessitates vaccines that offer broader protection from sarbecoviruses. By leveraging a viral-genome-informed computational method for selecting immune-optimized and structurally engineered antigens, here we show that a single antigen based on the receptor binding domain of the spike protein of sarbecoviruses elicits broad humoral responses against SARS-CoV-1, SARS-CoV-2, WIV16 and RaTG13 in mice, rabbits and guinea pigs. When administered as a DNA immunogen or by a vector based on a modified vaccinia virus Ankara, the optimized antigen induced vaccine protection from the Delta variant of SARS-CoV-2 in mice genetically engineered to express angiotensin-converting enzyme 2 and primed by a viral-vector vaccine (AZD1222) against SARS-CoV-2. A vaccine formulation incorporating mRNA coding for the optimized antigen further validated its broad immunogenicity. Vaccines that elicit broad immune responses across subgroups of coronaviruses may counteract the threat of zoonotic spillovers of betacoronaviruses.

The ideal vaccine against viral infections should elicit antibody responses that protect against divergent strains. Designing broadly protective vaccines against SARS-CoV-2 and other divergent viruses requires insight into the specific targets of cross-protective antibodies on the viral surface protein(s). However, unlike therapeutic monoclonal antibodies, the B-cell epitopes of vaccine-induced polyclonal antibody responses remain poorly defined. Here we show that, through the combination of neutralizing antibody functional responses with B-cell epitope mapping, it is possible to identify unique antibody targets associated with neutralization breadth. The polyclonal antibody profiles of SARS-CoV-2 index-strain-vaccinated rabbits that demonstrated a low, intermediate, or high neutralization efficiency of different SARS-CoV-2 variants of concern (VOCs) were distinctly different. Animals with an intermediate and high cross-neutralization of VOCs targeted fewer antigenic sites on the spike protein and targeted one particular epitope, subdomain 1 (SD1), situated outside the receptor binding domain (RBD). Our results indicate that a targeted functional antibody response and an additional focus on non-RBD epitopes could be effective for broad protection against different SARS-CoV-2 variants. We anticipate that the approach taken in this study can be applied to other viral vaccines for identifying future epitopes that confer cross-neutralizing antibody responses, and that our findings will inform a rational vaccine design for SARS-CoV-2.

The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. , ABSTRACT While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses – both X-ray and cryo-EM – revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability. IMPORTANCE The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.

Plasmodium ookinetes use an invasive apparatus to invade mosquito midguts, and tubulins are the major structural proteins of this apical complex. We examined the role of tubulins in malaria transmission to mosquitoes. Our results demonstrate that the rabbit polyclonal antibodies (pAb) against human α-tubulin significantly reduced the number of P. falciparum oocysts in Anopheles gambiae midguts, while rabbit pAb against human β-tubulin did not. Further studies showed that pAb, specifically against P. falciparum α-tubulin-1, also significantly limited P. falciparum transmission to mosquitoes. We also generated mouse monoclonal antibodies (mAb) using recombinant P. falciparum α-tubulin-1. Out of 16 mAb, two mAb, A3 and A16, blocked P. falciparum transmission with EC 50 of 12 μg/ml and 2.8 μg/ml. The epitopes of A3 and A16 were determined to be a conformational and linear sequence of EAREDLAALEKDYEE, respectively. To understand the mechanism of the antibody-blocking activity, we studied the accessibility of live ookinete α-tubulin-1 to antibodies and its interaction with mosquito midgut proteins. Immunofluorescent assays showed that pAb could bind to the apical complex of live ookinetes. Moreover, both ELISA and pull-down assays demonstrated that insect cell-expressed mosquito midgut protein, fibrinogen-related protein 1 (FREP1), interacts with P. falciparum α-tubulin-1. Since ookinete invasion is directional, we conclude that the interaction between Anopheles FREP1 protein and Plasmodium α -tubulin-1 anchors and orients the ookinete invasive apparatus towards the midgut PM and promotes the efficient parasite infection in the mosquito.