The core domain of hepatitis C virus glycoprotein E2 generates potent cross-neutralizing antibodies in guinea pigs.

A vaccine that prevents hepatitis C virus (HCV) infection is urgently needed to support an emerging global elimination program. However, vaccine development has been confounded because of HCV's high degree of antigenic variability and the preferential induction of type-specific immune responses with limited potency against heterologous viral strains and genotypes. We showed previously that deletion of the three variable regions from the E2 receptor-binding domain (Δ123) increases the ability of human broadly neutralizing antibodies (bNAbs) to inhibit E2-CD81 receptor interactions, suggesting improved bNAb epitope exposure. In this study, the immunogenicity of Δ123 was examined. We show that high-molecular-weight forms of Δ123 elicit distinct antibody specificities with potent and broad neutralizing activity against all seven HCV genotypes. Antibody competition studies revealed that immune sera raised to high-molecular-weight Δ123 was poly specific, given that it inhibited the binding of human bNAbs directed to three major neutralization epitopes on E2. By contrast, the immune sera raised to monomeric Δ123 predominantly blocked the binding of a non-neutralizing antibody to Δ123, while having reduced ability to block bNAb binding to E2, and neutralization was largely toward the homologous genotype. This increased ability of oligomeric Δ123 to generate bNAbs correlates with occlusion of the non-neutralizing face of E2 in this glycoprotein form. CONCLUSION: The results from this study reveal new information on the antigenic and immunogenic potential of E2-based immunogens and provide a pathway for the development of a simple, recombinant protein-based prophylactic vaccine for HCV with potential for universal protection. (Hepatology 2017;65:1117-1131).

Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region.

UNLABELLED: Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661 and E2661 lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion. IMPORTANCE: This study reveals conformational and antigenic differences between the Δ123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure.

ISCOMATRIX™ adjuvant reduces mucosal tolerance for effective pulmonary vaccination against influenza.

While most pathogens infect via mucosal surfaces, most current vaccines are delivered by injection. This situation remains despite awareness of the potential benefits of mucosal delivery for inducing protection against mucosa-infecting pathogens. A major obstacle to the development of such vaccines is the paucity of safe and effective adjuvants that induce mucosal responses in non-rodents. Previously we demonstrated in sheep the potency of pulmonary-delivered influenza ISCOMATRIX™ vaccine, which induces both mucosal and systemic immunity, even with low antigen doses. In the current study, lung pre-exposure to influenza antigen alone significantly reduced the immune response to subsequent pulmonary-delivered influenza ISCOMATRIX™ vaccine. A single dose of influenza antigen, delivered to the lung without exogenous adjuvant, upregulated IL-10 expression in bronchoalveolar lavage cells and FOXP3 expression in lung tissue, suggestive of induction of a regulatory T cell (Treg) response. However, this effect was inhibited by addition of ISCOMATRIX™ adjuvant. Moreover, effective pulmonary immunization with influenza ISCOMATRIX™ vaccine was associated with a depletion of Treg markers within lung tissues. Lung exposure to influenza antigen induced a localized mucosal tolerance that reduced the efficacy of subsequent influenza ISCOMATRIX™ vaccination. An important role of ISCOMATRIX™ adjuvant in pulmonary vaccination appears to be the depletion of Treg in lung tissues. Pulmonary vaccination remains capable of inducing a strong immune response against mucosal pathogens, but likely requires an adjuvant to overcome mucosal tolerance. ISCOMATRIX™ appears to have considerable potential as a mucosal adjuvant for use in humans, a major unmet need in mucosal vaccine development.

Helicobacter pylori thiolperoxidase as a protective antigen in single- and multi-component vaccines.

Helicobacter pylori is an important pathogen of the human stomach, and the development of a protective vaccine has been an enticing goal for many years. The H. pylori antioxidant enzymes superoxide dismutase (SOD) and catalase (KatA) have been shown to be protective as vaccine antigens in mice, demonstrating that the organism's antioxidant enzyme system is a fruitful target for vaccine development. The research described here demonstrates that an additional antioxidant enzyme, thiolperoxidase (Tpx), is effective as a prophylactic vaccine antigen via both systemic and mucosal routes. The functional relationship between SOD, KatA and Tpx also provided an opportunity to investigate synergistic or additive effects when the three antigens were used in combination. Although the antigens still provided equivalent protection when administered in combination, no additional protection was observed. Moreover a decrease in antibody titres to the individual antigens was observed when delivered in combination via the nasal route, though not when injected subcutaneously. The findings of this paper demonstrate that the antioxidant system of H. pylori presents a particularly rich resource for vaccine development.

Long-term antibody and immune memory response induced by pulmonary delivery of the influenza Iscomatrix vaccine.

Pulmonary delivery of an influenza Iscomatrix adjuvant vaccine induces a strong systemic and mucosal antibody response. Since an influenza vaccine needs to induce immunological memory that lasts at least 1 year for utility in humans, we examined the longevity of the immune response induced by such a pulmonary vaccination, with and without antigen challenge. Sheep were vaccinated in the deep lung with an influenza Iscomatrix vaccine, and serum and lung antibody levels were quantified for up to 1 year. The immune memory response to these vaccinations was determined following antigen challenge via lung delivery of influenza antigen at 6 months and 1 year postvaccination. Pulmonary vaccination of sheep with the influenza Iscomatrix vaccine induced antigen-specific antibodies in both sera and lungs that were detectable until 6 months postimmunization. Importantly, a memory recall response following antigenic challenge was detected at 12 months post-lung vaccination, including the induction of functional antibodies with hemagglutination inhibition activity. Pulmonary delivery of an influenza Iscomatrix vaccine induces a long-lived influenza virus-specific antibody and memory response of suitable length for annual vaccination against influenza.

Evaluation of superoxide dismutase from Helicobacter pylori as a protective vaccine antigen.

Helicobacter pylori, the major cause of gastric cancer, have mechanisms that allow colonization of the inhospitable gastric mucosa, including enzymes such as superoxide dismutase (SOD) which protect against reactive oxygen species. As SOD is essential for in vivo colonization, we theorized it might constitute a viable vaccine target. H. pylori SOD was expressed in E. coli and a purified recombinant protein used to vaccinate mice, prior to live H. pylori challenge. Partial protective immunity was induced, similar to that commonly observed with other antigens tested previously. This suggests SOD may have utility in a combination vaccine comprising several protective antigens.

Combined mucosal and systemic immunity following pulmonary delivery of ISCOMATRIX adjuvanted recombinant antigens.

Deep pulmonary delivery of an influenza ISCOMATRIX vaccine has previously been shown to induce a combined mucosal and systemic antibody response. To explore whether this combined response is influenced by intrinsic properties of the component antigen, we examined the efficacy of deep pulmonary delivery of ISCOMATRIX vaccines containing different recombinant antigens, specifically gB glycoprotein from cytomegalovirus and a fragment of catalase from Helicobacter pylori. Both these vaccines induced antigen-specific mucosal and systemic immunity, as well as antigen-specific proliferative cellular responses. Pulmonary immunisation with ISCOMATRIX vaccines may therefore be a generic way of inducing combined systemic and mucosal immunity.

Systemic immunization with unadjuvanted whole Helicobacter pylori protects mice against heterologous challenge.

BACKGROUND: Adjuvant-free vaccines have many benefits, including decreased cost and toxicity. We examined the protective effect of systemic vaccination with adjuvant-free formalin-fixed Helicobacter pylori or bacterial lysate and the ability of this vaccine to induce protection against heterologous challenge. MATERIALS AND METHODS: Mice were vaccinated subcutaneously with H. pylori 11637 lysate or formalin-fixed bacteria, with or without ISCOMATRIX adjuvant, then orally challenged with H. pylori SS1. Serum was taken prior to challenge to examine specific antibody levels induced by the vaccinations, and protection was assessed by colony-forming assay. RESULTS: Vaccination with H. pylori 11637 lysate or formalin-fixed bacteria delivered systemically induced significantly higher levels of Helicobacter-specific serum IgG than the control, unvaccinated group and orally vaccinated group. After heterologous challenge with H. pylori SS1, all vaccinated groups had significantly lower levels of colonization compared with unvaccinated, control mice, regardless of the addition of adjuvant or route of delivery. Protection induced by systemic vaccination with whole bacterial preparations, without the addition of adjuvants, was only associated with a mild cellular infiltration into the gastric mucosa, with no evidence of atrophy. CONCLUSIONS: Subcutaneous vaccination using unadjuvanted formalin-fixed H. pylori has the potential to be a simple, cost-effective approach to the development of a Helicobacter vaccine. Importantly, this vaccine was able to induce protection against heterologous challenge, a factor that would be crucial in any human Helicobacter vaccine. Further studies are required to determine mechanisms of protection and to improve protective ability.

Local immune responses following nasal delivery of an adjuvanted influenza vaccine.

A key barrier to producing effective nasal immunisations is the low efficiency of uptake of vaccines across the nasal mucosa. Using a recently developed cannulation system, we examined the antibody response induced by nasal immunisation with an ISCOMATRIX influenza vaccine. This showed for the first time, that following nasal vaccination, specific antibodies enter the circulation of primed animals via the draining lymphatics as a wave that peaks approximately 5-6 days after vaccination. These antibodies included some of the IgA isotype and possessed functional haemagglutination inhibition activity. These responses, though small, were induced using a very simple delivery system, emphasising the applicability of this cannulation model for evaluation of excipients and adjuvants aimed at improving intranasal vaccine efficacy.

Cervical cancer-causing human papillomaviruses have an alternative initiation site for the L1 protein.

All known sequences of the DNA encoding the major cervical cancer-causing human papillomavirus type 16 (HPV16) L1 capsid protein contain initiation codons which would allow translation to begin at either nucleotide 5559 or 5637. However the formation of virus-like particles (VLPs) only occurs efficiently when the initiation codon at nucleotide 5637 is used for in vitro expression studies. This knowledge, in concert with the fact that virions have not been observed in HPV16-infected epithelium, raises the notion that the major L1 translation product in this HPV type may be largely confined to initiation at nucleotide 5559. Sequence analysis of various HPV types associated with particular clinical outcomes has revealed that L1 sequences of the major cervical cancer-associated viruses generally possess the ability to encode a longer translation product whilst the non-cancer-causing viruses do not. Equally intriguing, the upstream initiation codon is always separated by 78 nucleotides from the initiation codon that produces L1 protein which efficiently assembles into VLPs. We speculate that the longer L1 protein could play a role in the development of cervical carcinoma and that HPVs with the potential to cause cervical cancer may be identified by the presence of an in-frame ATG situated 78 nucleotides upstream.

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX adjuvant in women with cervical intraepithelial neoplasia.

PURPOSE: Persistent infection of cervical epithelium with "high risk" human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX adjuvant (HPV16 Immunotherapeutic) for patients with CIN. EXPERIMENTAL DESIGN: Patients with CIN (n = 31) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. RESULTS: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. CONCLUSIONS: The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX adjuvant is safe and induces vaccine antigen specific cell mediated immunity.

ISCOMATRIX adjuvant: an adjuvant suitable for use in anticancer vaccines.

Human Papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are associated with cervical cancer development and progression and can therefore be used as target antigens for cancer immunotherapy. In this study we evaluated the immunogenicity in mice, of different vaccine formulations using recombinant HPV16 derived E6E7 or E7GST fusion proteins. When co-administered with ISCOMATRIX adjuvant, these E6E7 proteins consistently induced E7 specific CTL, in vivo tumor protection, antibody and DTH responses. ISCOMATRIX adjuvant has been developed for use in the formulation of novel human vaccines and has been evaluated for safety and toxicity in human trials. A formulation containing aluminum hydroxide (Al(OH)3) gave a lesser degree of E7 specific antibody, and no local E7 specific CTL response but similar DTH and tumor protection. These findings demonstrate the potential of ISCOMATRIX adjuvant to stimulate both cellular and humoral immune responses to endogenously processed target antigens, and hence is the preferred adjuvant when CTL responses are desirable.