Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.
Libraries based on an isolated human immunoglobulin G1 (IgG1) constant domain 2 (CH2) have been previously diversified by random mutagenesis. However, native isolated CH2 is not very stable and the generation of many mutations could lead to an increase in immunogenicity. Recently, we demonstrated that engineering an additional disulfide bond and removing seven N-terminal residues results in an engineered antibody domain (eAd) (m01s) with highly increased stability and enhanced binding to human neonatal Fc receptor (FcRn) (Gong et al, JBC, 2009 and 2011). We and others have also previously shown that grafting of the heavy chain complementarity region 3 (CDR-H3 (H3)) onto cognate positions of the variable domain leads to highly diversified libraries from which a number of binders to various antigens have been selected. However, grafting of H3s to non-cognate positions in constant domains results in additional residues at the junctions of H3s and the CH2 framework. Here we describe a new method based on multi-step PCR that allows the precise replacement of loop FG (no changes in its flanking sequences) by human H3s from another library. Using this method and limited mutagenesis of loops BC and DE we generated an eAd phage-displayed library. Panning of this library against an HIV-1 gp41 MPER peptide resulted in selection of a binder, m2a1, which neutralized HIV-1 isolates from different clades with modest activity and retained the m01s capability of binding to FcRn. This result provides a proof of concept that CH2-based antigen binders that also mimic to certain extent other functions of full-size antibodies (binding to FcRn) can be generated; we have previously hypothesized that such binders can be made and coined the term nanoantibodies (nAbs). Further studies in animal models and in humans will show how useful nAbs could be as therapeutics and diagnostics.
Many pathogens make use of antigenic variation as a way to evade the host immune response. A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. Recombination results in changes in the VlsE surface protein that prevent recognition by VlsE-specific antibodies in the infected host. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that B. burgdorferi may utilize a VlsE-mediated system for immune avoidance of its surface antigens. To address this, the requirement of VlsE for host reinfection by the Lyme disease pathogen was investigated. Host-adapted wild type and VlsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VlsE-deficient clone. Our results demonstrate that variable VlsE is necessary for reinfection by B. burgdorferi, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that VlsE prevents recognition of B. burgdorferi surface antigens from host antibodies. Overall, our findings represent a significant advance in our knowledge of immune evasion by B. burgdorferi, and provide insight to the possible mechanisms involved in VlsE-mediated immune avoidance.
Viral subunit vaccines often contain immunodominant non-neutralizing epitopes that divert host immune responses. These epitopes should be eliminated in vaccine design, but there is no reliable method for evaluating an epitope’s capacity to elicit neutralizing immune responses. Here we introduce a new concept ‘neutralizing immunogenicity index’ (NII) to evaluate an epitope’s neutralizing immunogenicity. To determine the NII, we mask the epitope with a glycan probe and then assess the epitope’s contribution to the vaccine’s overall neutralizing immunogenicity. As proof-of-concept, we measure the NII for different epitopes on an immunogen comprised of the receptor-binding domain from MERS coronavirus (MERS-CoV). Further, we design a variant form of this vaccine by masking an epitope that has a negative NII score. This engineered vaccine demonstrates significantly enhanced efficacy in protecting transgenic mice from lethal MERS-CoV challenge. Our study may guide the rational design of highly effective subunit vaccines to combat MERS-CoV and other life-threatening viruses.
Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4(+) T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens.
We have developed a cost-effective and portable graphene-enabled biosensor to detect Zika virus with a highly specific immobilized monoclonal antibody. Field Effect Biosensing (FEB) with monoclonal antibodies covalently linked to graphene enables real-time, quantitative detection of native Zika viral (ZIKV) antigens. The percent change in capacitance in response to doses of antigen (ZIKV NS1) coincides with levels of clinical significance with detection of antigen in buffer at concentrations as low as 450pM. Potential diagnostic applications were demonstrated by measuring Zika antigen in a simulated human serum. Selectivity was validated using Japanese Encephalitis NS1, a homologous and potentially cross-reactive viral antigen. Further, the graphene platform can simultaneously provide the advanced quantitative data of nonclinical biophysical kinetics tools, making it adaptable to both clinical research and possible diagnostic applications. The speed, sensitivity, and selectivity of this first-of-its-kind graphene-enabled Zika biosensor make it an ideal candidate for development as a medical diagnostic test.
Zika virus (ZIKV) was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of ZIKV in South America has led to widespread concern, with reports of neurological sequelae ranging from Guillain Barré syndrome to microcephaly. ZIKV infection has occurred in areas previously exposed to dengue virus (DENV), a flavivirus closely related to ZIKV. Here we investigated the serological cross-reaction between the two viruses. Plasma immune to DENV showed substantial cross-reaction to ZIKV and was able to drive antibody-dependent enhancement (ADE) of ZIKV infection. Using a panel of human monoclonal antibodies (mAbs) to DENV, we showed that most antibodies that reacted to DENV envelope protein also reacted to ZIKV. Antibodies to linear epitopes, including the immunodominant fusion-loop epitope, were able to bind ZIKV but were unable to neutralize the virus and instead promoted ADE. Our data indicate that immunity to DENV might drive greater ZIKV replication and have clear implications for disease pathogenesis and future vaccine programs for ZIKV and DENV.
Evaluation of the epitope specificities, location (systemic, mucosal) and effector function of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitude, epitope specificity, avidity and function of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4 mimetic miniprotein (gp140-M64U1) in rhesus macaques. Crosslinking of gp140 Env with M64U1 resulted in an earlier increase in both the magnitude and avidity of the IgG binding response compared to Env protein alone. Notably, binding IgG responses at an early time point correlated with Antibody Dependent Cellular Cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the crosslinked Env group compared to the Env group alone. In addition, the crosslinked Env group developed higher IgG responses against a linear epitope in the C1 gp120 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by crosslinking gp140 with the CD4 mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses that correlated with the rise of subsequent antibody-mediated antiviral functions.IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and the immune correlates analysis of this trial revealed a role for binding antibodies and antibody Fc mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that Env structural modifications that aim to mimic the CD4 bound conformation can result in earlier antibody elicitation, altered epitope specificity and increased antiviral function post immunization.
The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.
A major obstacle to a broadly neutralizing antibody (bnAb)-based HIV vaccine is the activation of appropriate B cell precursors. Germline-targeting immunogens must be capable of priming rare bnAb precursors in the physiological setting. We tested the ability of the VRC01-class bnAb germline-targeting immunogen eOD-GT8 60mer to activate appropriate precursors in mice transgenic for human immunoglobulin loci. Despite an average frequency of at most ~1 VRC01-class precursor per mouse, we found that at least 29% of singly-immunized mice produced a VRC01-class memory response, suggesting that priming generally succeeded when at least one precursor was present. The results demonstrate the feasibility of using germline targeting to prime specific and exceedingly rare bnAb precursor B cells within a human-like repertoire.