Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infections in horses. We hypothesized that high-avidity CTL specific for nonvariable epitopes might be associated with low viral load and minimal disease in EIAV-infected horses. To test this hypothesis, memory CTL (CTLm) responses were analyzed in two infected horses with high plasma viral loads and recurrent disease (progressors), and in two infected horses with low-to-undetectable viral loads and mild disease (nonprogressors). High-avidity CTLm in one progressor recognized an envelope gp90 epitope, and the data documented for the first time in EIAV that viral variation led to CTL escape. Each of the nonprogressors had high-to-moderate avidity CTLm directed against epitopes within Rev, including the nuclear export and nuclear localization domains. These results suggested that the epitope specificity of high- and moderate-avidity CTLm was an important determinant for disease outcome in the EIAV-infected horses examined.

Non-neutralising antibodies to the envelope glycoprotein are elicited during acute HIV-1 infection and are abundant throughout the course of disease progression. Although these antibodies appear to have negligible effects on HIV-1 infection when assayed in standard neutralisation assays, they have the potential to exert either inhibitory or enhancing effects through interactions with complement and/or Fc receptors. Here we report that non-neutralising antibodies produced early in response to HIV-1 infection can enhance viral infectivity.

Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.

Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Ig(mim2), CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4(+) T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection.

Antibodies of the VRC01 class neutralize HIV-1, arise in diverse HIV-1-infected donors, and are potential templates for an effective HIV-1 vaccine. However, the stochastic processes that generate repertoires in each individual of >10(12) antibodies make elicitation of specific antibodies uncertain. Here we determine the ontogeny of the VRC01 class by crystallography and next-generation sequencing. Despite antibody-sequence differences exceeding 50%, antibody-gp120 cocrystal structures reveal VRC01-class recognition to be remarkably similar. B cell transcripts indicate that VRC01-class antibodies require few specific genetic elements, suggesting that naive-B cells with VRC01-class features are generated regularly by recombination. Virtually all of these fail to mature, however, with only a few-likely one-ancestor B cell expanding to form a VRC01-class lineage in each donor. Developmental similarities in multiple donors thus reveal the generation of VRC01-class antibodies to be reproducible in principle, thereby providing a framework for attempts to elicit similar antibodies in the general population.

Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein-stabilized in the pre-fusion (pre-F) conformation by "DS-Cav1" mutations-elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These "head-only" immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting suggest these head-only RSV F immunogens, engineered to retain the pre-fusion conformation, may have advantages as candidate RSV vaccines.

The inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r=0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze-thaw cycles and storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections.

As predominant components of hemicelluloses in grasses, methylglucuroarabinoxylans (MeGAXn) are sources for the production of acidic xylooligosaccharides (U-XOS). Bacillus subtilis MR44, an engineered biocatalyst to secrete only the XynC xylanase and Axh43 arabinoxylan hydrolase is capable of processing MeGAXn to exclusively U-XOS. The present studies are directed at the explosion on direct alkaline extraction serving for production of U-XOS. Response Surface Methodology was used to optimize xylan extraction conditions on the sweet sorghum bagasse to achieve maximum hemicelluloses yield. The optimized condition was as follows: extraction time of 3.91 h, extraction temperature of 86.1°C, and NaOH concentration (w/w) of 12.33%. Crude xylan extracted with NaOH revealed a compositional analysis of xylose (79.0%), arabinose (5.3%), glucose (1.7%), lignin and ash (5.6%). After neutralization this xylan preparation supported growth of MR44, processing MeGAXn from sweet sorghum and accumulating U-XOS. The quality of U-XOS produced by MR44 using alkaline-treated sweet sorghum bagasse was comparable to that obtained from purified MeGAXn. Overall, the present study demonstrates that direct alkaline treatment of sweet sorghum bagasse is useful to improve the bioavailability of MeGAXn for MR44-mediated conversion to U-XOS with average degrees of polymerization of 11-12, providing alternative resources with applications in nutrition and human and veterinary medicine.

The canine transmissible venereal tumor (CTVT) is a clonally transmissible cancer that regresses spontaneously or after treatment with vincristine, but we know little about the regression mechanisms. We performed global transcriptional, methylation, and functional pathway analyses on serial biopsies of vincristine-treated CTVTs and found that regression occurs in sequential steps; activation of the innate immune system and host epithelial tissue remodeling followed by immune infiltration of the tumor, arrest in the cell cycle, and repair of tissue damage. We identified CCL5 as a possible driver of CTVT regression. Changes in gene expression are associated with methylation changes at specific intragenic sites. Our results underscore the critical role of host innate immunity in triggering cancer regression.

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 μg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

Antibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determine the structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three use VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy-chain N53I-enhancing binding and light-chain tyrosines recognizing F486RBD. Despite these similarities, class members bind both RBD-up and -down conformations of the spike, with a subset of antibodies using elongated CDRH3s to recognize glycan N343 on a neighboring RBD-a quaternary interaction accommodated by an increase in RBD separation of up to 12 Å. The VH1-2 antibody class, thus, uses modular recognition encoded by modular genetic elements to effect potent neutralization, with the VH-gene component specifying recognition of RBD and the CDRH3 component specifying quaternary interactions.

Numerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here, we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed that all seven antibodies target a common surface, bordered by glycans N17, N74, N122, and N149. This site-formed primarily by a mobile β-hairpin and several flexible loops-was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies appear to target a single supersite.

Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope.

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

The broadly neutralizing antibody (bNAb) CAP256-VRC26.25 has exceptional potency against HIV-1 and has been considered for clinical use. During the characterization and production of this bNAb, we observed several unusual features. First, the antibody appeared to adhere to pipette tips, requiring tips to be changed during serial dilution to accurately measure potency. Second, during production scale-up, proteolytic cleavage was discovered to target an extended heavy chain loop, which was attributed to a protease in spent medium from 2-week culture. To enable large scale production, we altered the site of cleavage via a single amino acid change, K100mA. The resultant antibody retained potency and breadth while avoiding protease cleavage. We also added the half-life extending mutation LS, which improved the in vivo persistence in animal models, but did not impact neutralization activity; we observed the same preservation of neutralization for bNAbs VRC01, N6, and PGDM1400 with LS on a 208-virus panel. The final engineered antibody, CAP256V2LS, retained the extraordinary neutralization potency of the parental antibody, had a favorable pharmacokinetic profile in animal models, and was negative in in vitro assessment of autoreactivity. CAP256V2LS has the requisite potency, developability and suitability for scale-up, allowing its advancement as a clinical candidate.

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

While several COVID-19 vaccines have been in use, more effective and durable vaccines are needed to combat the ongoing COVID-19 pandemic. Here, we report highly immunogenic self-assembling SARS-CoV-2 spike-HBsAg nanoparticles displaying a six-proline-stabilized WA1 (wild type, WT) spike S6P on a HBsAg core. These S6P-HBsAgs bound diverse domain-specific SARS-CoV-2 monoclonal antibodies. In mice with and without a HBV pre-vaccination, DNA immunization with S6P-HBsAgs elicited significantly more potent and durable neutralizing antibody (nAb) responses against diverse SARS-CoV-2 strains than that of soluble S2P or S6P, or full-length S2P with its coding sequence matching mRNA-1273. The nAb responses elicited by S6P-HBsAgs persisted substantially longer than by soluble S2P or S6P and appeared to be enhanced by HBsAg pre-exposure. These data show that genetic delivery of SARS-CoV-2 S6P-HBsAg nanoparticles can elicit greater and more durable nAb responses than non-nanoparticle forms of stabilized spike. Our findings highlight the potential of S6P-HBsAgs as next generation genetic vaccine candidates against SARS-CoV-2.