Influenza Hemagglutinin Head Domain Mimicry by Rational Design.

Despite diligent vaccination efforts, influenza virus infection remains a major cause for respiratory-related illness across the globe. The less-than-optimal immunity conferred by the currently prescribed seasonal vaccines and protracted production times warrant the development of novel vaccines. Induction of an epitope-focused antibody response targeting known neutralization epitopes is a viable strategy to enhance the breadth of protection against rapidly evolving infectious viruses. We report the development of a design framework to mimic the hemagglutinin (HA) head fragment of H1-subtype viruses by delineating the interaction network of invariant residues lining the receptor binding site (RBS); a site targeted by cross-reactive neutralizing antibodies. The incorporation of multiple sequence alignment information in our algorithm to fix the construct termini and engineer rational mutations facilitates the facile extension of the design to heterologous (subtype-specific) influenza strains. We evaluated our design protocol by generating head fragments from divergent influenza A H1N1 A/Puerto Rico/8/34 and pH1N1 A/California/07/2009 strains that share a sequence identity of only 74.4% within the HA1 subunit. The designed immunogens exhibited characteristics of a well-ordered protein, and bound conformation-specific RBS targeting antibodies with high affinity, a desirable feature for putative vaccine candidates. Additionally, the bacterial expression of these immunogens provides a low-cost, rapidly scalable alternative.

Stalking influenza by vaccination with pre-fusion headless HA mini-stem.

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies induced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.

Generation and Characterization of Monoclonal Antibodies Specific to Avian Influenza H5N1 Hemagglutinin Protein.

Highly pathogenic avian influenza (HPAI) H5N1 virus has in the past breached the species barrier from infected domestic poultry to humans in close contact. Although human-to-human transmission has previously not been reported, HPAI H5N1 virus has pandemic potential owing to gain of function mutation(s) and/or genetic reassortment with human influenza A viruses. Monoclonal antibodies (MAbs) have been used for diagnosis as well as specific therapeutic candidates in several disease conditions including viral infections in humans. In this study, we describe the preliminary characterization of four murine MAbs developed against recombinant hemagglutinin (rHA) protein of avian H5N1 A/turkey/Turkey/1/2005 virus that are either highly specific or broadly reactive against HA from other H5N1 subtype viruses, such as A/Hong Kong/213/03, A/Common magpie/Hong Kong/2256/2006, and A/Barheaded goose/Quinghai/14/2008. The antibody binding is specific to H5N1 HAs, as none of the antibodies bound H1N1, H2N2, H3N2, or B/Brisbane/60/2008 HAs. Out of the four MAbs, one of them (MA-7) also reacted weakly with the rHA protein of H7N9 A/Anhui/1/2013. All four MAbs bound H5 HA (A/turkey/Turkey/1/2005) with high affinity with an equilibrium dissociation constant (KD) ranging between 0.05 and 10.30 nM. One of the MAbs (MA-1) also showed hemagglutination inhibition activity (HI titer; 31.25 µg/mL) against the homologous A/turkey/Turkey/1/2005 H5N1 virus. These antibodies may be useful in developing diagnostic tools for detection of influenza H5N1 virus infection.

Hemagglutinin Sequence Conservation Guided Stem Immunogen Design from Influenza A H3 Subtype.

Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential "universal" vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon". These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

Immunogen design for HIV-1 and influenza.

Vaccines provide the most cost effective defense against pathogens. Although vaccines have been designed for a number of viral diseases, a vaccine against HIV-1 still remains elusive. In contrast, while there are excellent influenza vaccines, these need to be changed every few years because of antigenic drift and shift. The recent discovery of a large number of broadly neutralizing antibodies (bNAbs) and structural characterization of the conserved epitopes targeted by them presents an opportunity for structure based HIV-1 and influenza A vaccine design. We discuss strategies to design immunogens either targeting a particular antigenic region or focusing on native structure stabilization. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

In vitro and in vivo characterization of designed immunogens derived from the CD-helix of the stem of influenza hemagglutinin.

The conserved "stem" domain of influenza virus hemagglutinin (HA) is a target for broadly neutralizing antibodies and a potential vaccine antigen for induction of hetero-subtypic protection. The epitope of 12D1, a previously reported bnAb neutralizing several H3 subtype influenza strains, was putatively mapped to residues 76-106 of the CD-helix, also referred to as long alpha helix (LAH) of the HA stem. A peptide derivative consisting of wt-LAH residues 76-130 conjugated to keyhole limpet hemocyanin was previously shown to confer robust protection in mice against challenge with influenza strains of subtypes H3, H1, and H5 which motivated the present study. We report the design of multiple peptide derivatives of LAH with or without heterologous trimerization sequences and show that several of these are better folded than wt-LAH. However, in contrast to the previous study immunization of mice with wt-LAH resulted in negligible protection against a lethal homologous virus challenge, while some of the newly designed immunogens could confer weak protection. Combined with structural analysis of HA, our data suggest that in addition to LAH, other regions of HA are likely to significantly contribute to the epitope for 12D1 and will be required to elicit robust protection. In addition, a dynamic, flexible conformation of isolated LAH peptide may be required for eliciting a functional anti-viral response.