Modification of Adenovirus vaccine vector-induced immune responses by expression of a signalling molecule.

Adenoviral vectors are being developed as vaccines against infectious agents and tumour-associated antigens, because of their ability to induce cellular immunity. However, the protection afforded in animal models has not easily translated into primates and clinical trials, underlying the need for improving adenoviral vaccines-induced immunogenicity. A Toll-like receptor signalling molecule, TRAM, was assessed for its ability to modify the immune responses induced by an adenovirus-based vaccine. Different adenovirus vectors either expressing TRAM alone or co-expressing TRAM along with a model antigen were constructed. The modification of T-cell and antibody responses induced by TRAM was assessed in vivo in mice and in primates. Co-expression of TRAM and an antigen from adenoviruses increased the transgene-specific CD8+ T cell responses in mice. Similar effects were seen when a TRAM expressing virus was co-administered with the antigen-expressing adenovirus. However, in primate studies, co-administration of a TRAM expressing adenovirus with a vaccine expressing the ME-TRAP malaria antigen had no significant effect on the immune responses. While these results support the idea that modification of innate immune signalling by genetic vectors modifies immunogenicity, they also emphasise the difficulty in generalising results from rodents into primates, where the regulatory pathway may be different to that in mice.

4-1BBL enhances CD8+ T cell responses induced by vectored vaccines in mice but fails to improve immunogenicity in rhesus macaques.

T cells play a central role in the immune response to many of the world's major infectious diseases. In this study we investigated the tumour necrosis factor receptor superfamily costimulatory molecule, 4-1BBL (CD137L, TNFSF9), for its ability to increase T cell immunogenicity induced by a variety of recombinant vectored vaccines. To efficiently test this hypothesis, we assessed a number of promoters and developed a stable bi-cistronic vector expressing both the antigen and adjuvant. Co-expression of 4-1BBL, together with our model antigen TIP, was shown to increase the frequency of murine antigen-specific IFN-γ secreting CD8(+) T cells in three vector platforms examined. Enhancement of the response was not limited by co-expression with the antigen, as an increase in CD8(+) immunogenicity was also observed by co-administration of two vectors each expressing only the antigen or adjuvant. However, when this regimen was tested in non-human primates using a clinical malaria vaccine candidate, no adjuvant effect of 4-1BBL was observed limiting its potential use as a single adjuvant for translation into a clinical vaccine.

Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5.

There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5-based malaria prevention efforts.

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice.

BACKGROUND: Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. METHODS: Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. RESULTS: A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. CONCLUSIONS: Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens.

No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50) values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates.

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.

Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass.

Live recombinant viral vectors based on adenoviruses and poxviruses are among the most promising platforms for development of new vaccines against diseases such as malaria, tuberculosis, and HIV-AIDS. Vaccines based on live viruses must remain infectious to be effective, so therefore need continuous refrigeration to maintain stability and viability, a requirement that can be costly and difficult, especially in developing countries. The sugars sucrose and trehalose are commonly used as stabilizing agents and cryoprotectants for biological products. Here, we have exploited the ability of these sugars to vitrify on desiccation to develop a thermostabilization technique for live viral vaccine vectors. By slowly drying vaccines suspended in solutions of these disaccharide stabilizers onto a filter-like support membrane at ambient temperature, an ultrathin glass is deposited on the fibers of the inert matrix. Immobilization of two recombinant vaccine vectors-E1/E3-deleted human adenovirus type 5 and modified vaccinia virus Ankara-in this glass on the membranes enabled complete recovery of viral titer and immunogenicity after storage at up to 45 degrees C for 6 months and even longer with minimal losses. Furthermore, the membrane carrying the stabilized vaccine can be incorporated into a holder attached to a syringe for almost simultaneous reconstitution and injection at point of use. The technology may potentially be developed for the deployment of viral vector-based biopharmaceuticals in resource-poor settings.

Expression of tak1 and tram induces synergistic pro-inflammatory signalling and adjuvants DNA vaccines.

Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants.

Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA).

The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC) in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20). The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA), now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+) T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+) T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins), in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006). In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+) T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant vaccines based on a clinically deployable viral vector.

Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches.

Chimeric 101F (ch101F) is a mouse-human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422-438 spanning antigenic site IV, V, VI was analysed. Residues 423-436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.

Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins.

Alpha/beta interferons (IFN-alpha/beta) are not only a powerful first line of defense against pathogens but also have potent immunomodulatory activities. Many viruses have developed mechanisms of subverting the IFN system to enhance their virulence. Previous studies have demonstrated that the nonstructural (NS) genes of bovine respiratory syncytial virus (BRSV) counteract the antiviral effects of IFN-alpha/beta. Here we demonstrate that, in contrast to wild-type BRSVs, recombinant BRSVs (rBRSVs) lacking the NS proteins, and those lacking NS2 in particular, are strong inducers of IFN-alpha/beta in bovine nasal fibroblasts and bronchoalveolar macrophages. Furthermore, whereas the NS deletion mutants replicated to wild-type rBRSV levels in cells lacking a functional IFN-alpha/beta system, their replication was severely attenuated in IFN-competent cells and in young calves. These results suggest that the NS proteins block the induction of IFN-alpha/beta gene expression and thereby increase the virulence of BRSV. Despite their poor replication in the respiratory tract of young calves, prior infection with virus lacking either the NS1 or the NS2 protein induced serum antibodies and protection against challenge with virulent BRSV. The greater level of protection induced by the NS2, than by the NS1, deletion mutant, was associated with higher BRSV-specific antibody titers and greater priming of BRSV-specific, IFN-gamma-producing CD4(+) T cells. Since there were no detectable differences in the ability of these mutants to replicate in the bovine respiratory tract, the greater immunogenicity of the NS2 deletion mutant may be associated with the greater ability of this virus to induce IFN-alpha/beta.

Recognition of bovine respiratory syncytial virus proteins by bovine CD8+ T lymphocytes.

CD8+ T lymphocytes play a major role in the clearance of bovine respiratory syncytial virus (BRSV), an important respiratory pathogen of young calves that shares many of the epidemiological and pathological features of human respiratory syncytial virus (HRSV) in infants. Recombinant vaccinia virus (rVV) and recombinant fowlpox virus (rFPV), expressing individual BRSV proteins, were used to demonstrate that the F, N and M2 proteins were the major antigens recognized by bovine CD8+ T cells in major histocompatibility complex (MHC)-defined cattle. BRSV protein recognition by CD8+ T cells was analysed using cytotoxic T lymphocyte (CTL) assays or by the production of interferon-gamma (IFN-gamma) following restimulation with BRSV proteins. Strong recognition of the G protein by CD8+ T cells was observed in cattle that had been vaccinated with rVV expressing this protein and subsequently challenged with BRSV. Although there is variation in the number of expressed MHC genes in cattle with different class I haplotypes, this did not appear to influence BRSV protein recognition by CD8+ T cells. Knowledge of the antigenic specificity of BRSV-specific CD8+ T cells will facilitate the qualitative and quantitative analysis of BRSV-specific CD8+ T-cell memory in cattle and help to ensure that potential vaccines induce a qualitatively appropriate CD8+ T-cell response.