Sally P. Mossman

Cross-Protection against Lethal H5N1 Challenge in Ferrets with an Adjuvanted Pandemic Influenza Vaccine

Background Unprecedented spread between birds and mammals of highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtype has resulted in hundreds of human infections with a high fatality rate. This has highlighted the urgent need for the development of H5N1 vaccines that can be produced rapidly and in sufficient quantities. Potential pandemic inactivated vaccines will ideally induce substantial intra-subtypic cross-protection in humans to warrant the option of use, either prior to or just after the start of a pandemic outbreak. In the present study, we evaluated a split H5N1 A/H5N1/Vietnam/1194/04, clade 1 candidate vaccine, adjuvanted with a proprietary oil-in- water emulsion based Adjuvant System proven to be well-tolerated and highly immunogenic in the human (Leroux-Roels et al. (2007) The Lancet 370:580–589), for its ability to induce intra-subtypic cross-protection against clade 2 H5N1/A/Indonesia/5/05 challenge in ferrets. Methodology and Principal Findings All ferrets in control groups receiving non-adjuvanted vaccine or adjuvant alone failed to develop specific or cross-reactive neutralizing antibodies and all died or had to be euthanized within four days of virus challenge. Two doses of adjuvanted split H5N1 vaccine containing ≥1.7 µg HA induced neutralizing antibodies in the majority of ferrets to both clade 1 (17/23 (74%) responders) and clade 2 viruses (14/23 (61%) responders), and 96% (22/23) of vaccinees survived the lethal challenge. Furthermore lung virus loads and viral shedding in the upper respiratory tract were reduced in vaccinated animals relative to controls suggesting that vaccination might also confer a reduced risk of viral transmission. Conclusion These protection data in a stringent challenge model in association with an excellent clinical profile highlight the potential of this adjuvanted H5N1 candidate vaccine as an effective tool in pandemic preparedness.

Protection from pathogenic SIV challenge using multigenic DNA vaccines

To assess DNA immunization as a strategy for protecting against HIV infection in humans, we utilized SIVmne infection of Macaca fascicularis as a vaccine challenge model with moderate pathogenic potential. We compared the efficacy of DNA immunization alone and in combination with subunit protein boosts. All of the structural and regulatory genes of SIVmne clone 8 were cloned into mammalian expression vectors under the control of the CMV IE-1 promoter. Eight M. fascicularis were immunized twice with 3 mg of plasmid DNA divided between two sites; intramuscular and intradermal. Four primed macaques received a further two DNA immunizations at weeks 16-36, while the second group of four were boosted with 250 microg recombinant gp160 plus 250 microg recombinant Gag-Pol particles formulated in MF-59 adjuvant. Half of the controls received four immunizations of vector DNA; half received two vector DNA and two adjuvant immunizations. As expected, humoral immune responses were stronger in the macaques receiving subunit boosts, but responses were sustained in both groups. Significant neutralizing antibody titers to SIVmne were detected in one of the subunit-boosted animals and in none of the DNA-only animals prior to challenge. T-cell proliferative responses to gp160 and to Gag were detected in all immunized animals after three immunizations, and these responses increased after four immunizations. Cytokine profiles in PHA-stimulated PBMC taken on the day of challenge showed trends toward Thl responses in 2/4 macaques in the DNA vaccinated group and in 1/4 of the DNA plus subunit vaccinated macaques; Th2 responses in 3/4 DNA plus subunit-immunized macaques; and Th0 responses in 4/4 controls. In bulk CTL culture, SIV specific lysis was low or undetectable, even after four immunizations. However, stable SIV Gag-Pol- and env-specific T-cell clones (CD3+ CD8+) were isolated after only two DNA immunizations, and Gag-Pol- and Nef-specific CTL lines were isolated on the day of challenge. All animals were challenged at week 38 with SIVmne uncloned stock by the intrarectal route. Based on antibody anamnestic responses (western, ELISA, and neutralizing antibodies) and virus detection methods (co-culture of PBMC and LNMC, nested set PCR- of DNA from PBMC and LNMC, and plasma QC-PCR), there were major differences between the groups in the challenge outcome. Surprisingly, sustained low virus loads were observed only in the DNA group, suggesting that four immunizations with DNA only elicited more effective immune responses than two DNA primes combined with two protein boosts. Multigenic DNA vaccines such as these, bearing all structural and regulatory genes, show significant promise and may be a safe alternative to live-attenuated vaccines.

Hepatitis B vaccine effectiveness in the face of global HBV genotype diversity

Recombinant hepatitis B vaccines are of the A2 genotype; one of ten known genotypes whose distribution varies globally. Reports of rare HBV infections in blood donors with an imbalance of non-A2 genotype HBV in vaccinated subjects have raised questions about the cross-protection afforded by HBV-A2 vaccines. Infections in HBV vaccinees were asymptomatic and transient, indicating that vaccination prevented clinical disease. Preclinical data demonstrate cross-reactivity and cross-protection by A2 vaccines against non-A2 HBV genotypes. Substantial improvements in HBV control have been demonstrated in countries with diverse genotype distribution that have introduced universal childhood HBV vaccination programs. Available data show that current HBV-A2 vaccines are highly effective in preventing infections and clinical disease caused by all known HBV genotypes.

Protective Immunity to SIV Challenge Elicited by Vaccination of Macaques with Multigenic DNA Vaccines Producing Virus-Like Particles

We utilized SIV(mne) infection of Macaca fascicularis to assess the efficacy of DNA vaccination alone, and as a priming agent in combination with subunit protein boosts. All SIV(mne) structural and regulatory genes were expressed using the human cytomegalovirus Immediate Early-1 promoter in plasmids that directed the formation of virus-like particles in vitro. Macaques (n = 4) were immunized intradermally and intramuscularly four times over 36 weeks with 3 mg plasmid DNA. A second group (n = 4) received two DNA priming inoculations followed by two intramuscular boosts consisting of 250 microg recombinant Env gp160 and 250 microg recombinant Gag-Pol particles in MF-59 adjuvant. These regimens elicited modest cellular immunity prior to challenge. Humoral immune responses to Env gp160 were elicited and sustained by both vaccine protocols, and as expected antibody titers were higher in the protein subunit-boosted animals. Neutralizing antibodies prior to challenge were measurable in two of four subunit-boosted macaques. The two vaccine regimens elicited comparable helper T cell responses at the time of challenge. Vaccinees and mock-immunized controls (n = 4) were challenged intrarectally at week 38 with uncloned SIV(mne). Following challenge all macaques became infected, but both vaccine regimens resulted in reduced peak virus loads (p = 0.07) and significantly improved maintenance of peripheral CD4(+) T cell counts postchallenge (p = 0.007, DNA alone and p = 0.01, all vaccinees). There was no significant difference between the two vaccine groups in levels of plasma viremia or maintenance of CD4(+) T cell counts postchallenge.

Glycoprotein B (gB) vaccines adjuvanted with AS01 or AS02 protect female guinea pigs against cytomegalovirus (CMV) viremia and offspring mortality in a CMV-challenge model

The transmission of cytomegalovirus (CMV) from mother to fetus can give rise to severe neurodevelopment defects in newborns. One strategy to prevent these congenital defects is prophylactic vaccination in young women. A candidate vaccine antigen is glycoprotein B (gB). This antigen is abundant on the virion surface and is a major target of neutralization responses in human infections. Here, we have evaluated in a challenge model of congenital guinea pig CMV (GPCMV) infection, GPCMV-gB vaccines formulated with the clinically relevant Adjuvant Systems AS01B and AS02V, or with Freunds adjuvant (FA). Fifty-two GPCMV-seronegative female guinea pigs were administered three vaccine doses before being mated. GPCMV-challenge was performed at Day 45 of pregnancy (of an estimated 65 day gestation). Pup mortality rates in the gB/AS01B, gB/AS02V, and gB/FA groups were 24% (8/34), 10% (4/39) and 36% (12/33), respectively, and in the unvaccinated control group was 65% (37/57). Hence, efficacies against pup mortality were estimated at 64%, 84% and 44% for gB/AS01B (p<0.001), gB/AS02V (p<0.001) and gB/FA (p=0.014), respectively. Efficacies against GPCMV viremia (i.e. DNAemia, detected by PCR) were estimated at 88%, 68% and 25% for the same vaccines, respectively, but were only significant for gB/AS01B (p<0.001), and gB/AS02V (p=0.002). In dams with viremia, viral load was approximately 6-fold lower with vaccination than without. All vaccines were highly immunogenic after two and three doses. In light of these results and of other results of AS01-adjuvanted vaccines in clinical development, vaccine immunogenicity was further explored using human CMV-derived gB antigen adjuvanted with either AS01B or the related formulation AS01E. Both adjuvanted vaccines were highly immunogenic after two doses, in contrast to the lower immunogenicity of the unadjuvanted vaccine. In conclusion, the protective efficacy and immunogenicity of adjuvanted vaccines in this guinea pig model are supportive of investigating gB/AS01 and gB/AS02 in the clinic.

Pandemic H1N1 vaccine requires the use of an adjuvant to protect against challenge in naïve ferrets

In the context of an A/H1N1 influenza pandemic situation, this study demonstrates that heterologous vaccination with an AS03-adjuvanted 2008/2009 seasonal trivalent and pandemic H5N1 monovalent split vaccine conferred partial protection in influenza-naïve ferrets after challenge with the influenza pandemic H1N1 A/The Netherlands/602/09 virus. Further, unlike saline control and non-adjuvanted vaccine, it was shown that immunization of naïve ferrets with an AS03-adjuvanted pandemic H1N1 A/California/7/09 influenza split vaccine induced increased antibody response and enhanced protection against the challenge strain, including significant reduction in viral shedding in the upper respiratory tract and reduced lung pathology post-challenge. These results show the need for vaccination with the adjuvanted vaccine to fully protect against viral replication and influenza disease in unprimed ferrets.

Macaque blood-derived antigen-presenting cells elicit SIV-specific immune responses

Natural blood‐borne antigen‐presenting cells (APCs) were tested for their ability to augment antigen presentation for SIV vaccines. Fibrocytes and monocyte‐derived dendritic cells (DCs) were isolated from multiple Macaca fascicularis. Macaque fibrocytes displayed the characteristic cellular morphology and stained positive for CD34 and collagen, as observed in human and murine fibrocytes. Macaque DCs were generated from monocytes by culturing in granulocyte–macrophage colony stimulating factor and interleukin‐4 (IL‐4). Two days after maturation, cells were enriched for the DC marker CD83. Fibrocytes and DCs were each transfected with green fluorescence protein expression plasmids or DNA expression vectors encoding all of the SIVmne structural and regulatory genes. Autologous DCs were re‐infused into macaques subcutaneously (sc) following transfection; mixing with recombinant SIV antigens or inactivated whole SIV in vitro; or mock‐treatment. Autologous monocyte‐derived DCs pulsed with whole inactivated SIV were re‐infused and elicited cellular and/or humoral responses in vivo in eight of ten vaccinated macaques.

Immunization against SIVmne in macaques using multigenic DNA vaccines.

Abstract: All structural and regulatory genes of SIVmne were cloned into mammalian expression vectors to optimize expression in vitro and immunogenicity in mice. Macaca fascicularis were immunized four times with plasmid DNA (n = 4), or two DNA priming inoculations followed by two boosts of recombinant gp160 plus Gag‐Pol particles (n = 4). Following intrarectal challenge with SIVmne, all macaques became infected. Three monkeys immunized with DNA alone maintained low plasma virus loads by 1 year post‐challenge; the fourth exhibited high virus loads and significant CD4 + cell decline. Two of the DNA plus boost and three control macaques had high virus loads and associated CD4 + cell decline. Both vaccine protocols elicited antibodies and comparable helper T‐cell proliferative responses to gp160. Cytokine mRNA levels in activated peripheral blood mononuclear cells (PBMC) taken at time of challenge suggested a dominant T helper (Th) 1 state in three DNA‐immunized and one protein‐boosted macaque, which correlated with low virus loads and high CD4 + cell counts post‐challenge.

In vivo cell and tissue tropism of SIVsmmPBj14-bcl.3.

To gain insight into the unique pathogenicity of simian immunodeficiency virus (SIV) variant PBj14, which produces an acutely lethal enteropathic syndrome in infected pigtail macaques, we investigated the cell and tissue tropisms of a highly pathogenic biologic clone (bcl.3) of SIVsmmPBj14. To compare the relative amount of viral antigen in lymphoid organs of infected macaques we used an objective semiquantitative immunohistochemistry (sQIHC) assay. We found that in all animals viral antigen load was greater in alimentary-associated lymphoid tissues (gut-associated lymphoid tissue [GALT], tonsil, mesenteric and retropharyngeal lymph nodes) than in non-alimentary-associated lymphoid tissues (spleen, thymus, inguinal and axillary lymph nodes). Moreover, in six of nine animals examined, virus load in GALT was greater than that in any other lymphoid tissue. To determine whether the acute pathogenicity and prolific replication of SIVsmmPBj14 might be explained by a broader in vivo cell tropism than is typical of SIVs, we used cell subset separation and nested PCR. We found that the primary target cells in mesenteric lymph node for SIVsmmPBj14 were CD4+ T lymphocytes. However, the virus also infected macrophages, as well as CD8+ T cells and B cells, albeit at low frequencies. These results suggest that alimentary lymphoid tissue localization rather than unusual cell phenotype tropism distinguishes the singular pathogenesis of SIVsmmPBj14.

Longevity of the protective immune response induced after vaccination with one or two doses of AS03A-adjuvanted split H5N1 vaccine in ferrets

It is crucial that a safe and effective pandemic vaccine be rapidly available to combat a new pandemic threat. In this study we investigated the magnitude and persistence of the protective efficacy induced by one or two doses (3.75 μg HA/dose) of AS03(A)-adjuvanted H5N1 A/Indonesia/5/05 split vaccine in a lethal ferret challenge model. All ferrets that received at least one dose of adjuvanted vaccine 4 weeks before homologous challenge survived and showed reduced or undetectable virus replication in the lungs and the upper airways. Ferrets receiving two doses of adjuvanted vaccine 19 and 16 weeks before the challenge also showed high level of protection from replication in the lungs and the upper airways, albeit with only 83% survival. Animals in the control groups (non-adjuvanted vaccine or saline) and animals immunized with one dose of adjuvanted vaccine administered 10 or 16 weeks before challenge showed only 17-33% survival rate after challenge. In conclusion, our observations support the possibility that a single dose of AS03(A)-adjuvanted H5N1 split vaccine can offer a rapid and short term but partial protection against disease. A second dose of the adjuvanted vaccine, which can be given with a flexible injection schedule, was shown to be essential to induce appreciable levels of antibodies and long-term protection.