Immune responses associated with homologous protection conferred by commercial vaccines for control of avian pathogenic Escherichia coli in turkeys.

Avian pathogenic Escherichia coli (APEC) infections are a serious impediment to sustainable poultry production worldwide. Licensed vaccines are available, but the immunological basis of protection is ill-defined and a need exists to extend cross-serotype efficacy. Here, we analysed innate and adaptive responses induced by commercial vaccines in turkeys. Both a live-attenuated APEC O78 ΔaroA vaccine (Poulvac® E. coli) and a formalin-inactivated APEC O78 bacterin conferred significant protection against homologous intra-airsac challenge in a model of acute colibacillosis. Analysis of expression levels of signature cytokine mRNAs indicated that both vaccines induced a predominantly Th2 response in the spleen. Both vaccines resulted in increased levels of serum O78-specific IgY detected by ELISA and significant splenocyte recall responses to soluble APEC antigens at post-vaccination and post-challenge periods. Supplementing a non-adjuvanted inactivated vaccine with Th2-biasing (Titermax® Gold or aluminium hydroxide) or Th1-biasing (CASAC or CpG motifs) adjuvants, suggested that Th2-biasing adjuvants may give more protection. However, all adjuvants tested augmented humoral responses and protection relative to controls. Our data highlight the importance of both cell-mediated and antibody responses in APEC vaccine-mediated protection toward the control of a key avian endemic disease.

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.

CD8+ T effector memory cells protect against liver-stage malaria.

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.

The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus.

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.

Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity.

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8(+) T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8(+) T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8(+) response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.

Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms.

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.

Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1.

BACKGROUND: Influenza A viruses cause occasional pandemics and frequent epidemics. Licensed influenza vaccines that induce high antibody titers to the highly polymorphic viral surface antigen hemagglutinin must be re-formulated and readministered annually. A vaccine providing protective immunity to the highly conserved internal antigens could provide longer-lasting protection against multiple influenza subtypes. METHODS: We prepared a Modified Vaccinia virus Ankara (MVA) vector encoding nucleoprotein and matrix protein 1 (MVA-NP+M1) and conducted a phase I clinical trial in healthy adults. RESULTS: The vaccine was generally safe and well tolerated, with significantly fewer local side effects after intramuscular rather than intradermal administration. Systemic side effects increased at the higher dose in both frequency and severity, with 5 out of 8 volunteers experiencing severe nausea/vomiting, malaise, or rigors. Ex vivo T-cell responses to NP and M1 measured by IFN-γ ELISPOT assay were significantly increased after vaccination (prevaccination median of 123 spot-forming units/million peripheral blood mononuclear cells, postvaccination peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), and the majority of the antigen-specific T cells were CD8(+). CONCLUSIONS: We conclude that the vaccine was both safe and remarkably immunogenic, leading to frequencies of responding T cells that appear to be much higher than those induced by any other influenza vaccination approach. Further studies will be required to find the optimum dose and to assess whether the increased T-cell response to conserved influenza proteins results in protection from influenza disease.

TLR Agonists as Vaccine Adjuvants Targeting Cancer and Infectious Diseases.

Modern vaccines have largely shifted from using whole, killed or attenuated pathogens to being based on subunit components. Since this diminishes immunogenicity, vaccine adjuvants that enhance the immune response to purified antigens are critically needed. Further advantages of adjuvants include dose sparing, increased vaccine efficacy in immunocompromised individuals and the potential to protect against highly variable pathogens by broadening the immune response. Due to their ability to link the innate with the adaptive immune response, Toll-like receptor (TLR) agonists are highly promising as adjuvants in vaccines against life-threatening and complex diseases such as cancer, AIDS and malaria. TLRs are transmembrane receptors, which are predominantly expressed by innate immune cells. They can be classified into cell surface (TLR1, TLR2, TLR4, TLR5, TLR6) and intracellular TLRs (TLR3, TLR7, TLR8, TLR9), expressed on endosomal membranes. Besides a transmembrane domain, each TLR possesses a leucine-rich repeat (LRR) segment that mediates PAMP/DAMP recognition and a TIR domain that delivers the downstream signal transduction and initiates an inflammatory response. Thus, TLRs are excellent targets for adjuvants to provide a "danger" signal to induce an effective immune response that leads to long-lasting protection. The present review will elaborate on applications of TLR ligands as vaccine adjuvants and immunotherapeutic agents, with a focus on clinically relevant adjuvants.

Neutrophilia, lymphopenia and myeloid dysfunction: a living review of the quantitative changes to innate and adaptive immune cells which define COVID-19 pathology.

Destabilization of balanced immune cell numbers and frequencies is a common feature of viral infections. This occurs due to, and further enhances, viral immune evasion and survival. Since the discovery of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which manifests in coronavirus disease 2019 (COVID-19), a great number of studies have described the association between this virus and pathologically increased or decreased immune cell counts. In this review, we consider the absolute and relative changes to innate and adaptive immune cell numbers, in COVID-19. In severe disease particularly, neutrophils are increased, which can lead to inflammation and tissue damage. Dysregulation of other granulocytes, basophils and eosinophils represents an unusual COVID-19 phenomenon. Contrastingly, the impact on the different types of monocytes leans more strongly to an altered phenotype, e.g. HLA-DR expression, rather than numerical changes. However, it is the adaptive immune response that bears the most profound impact of SARS-CoV-2 infection. T cell lymphopenia correlates with increased risk of intensive care unit admission and death; therefore, this parameter is particularly important for clinical decision-making. Mild and severe diseases differ in the rate of immune cell counts returning to normal levels post disease. Tracking the recovery trajectories of various immune cell counts may also have implications for long-term COVID-19 monitoring. This review represents a snapshot of our current knowledge, showing that much has been achieved in a short period of time. Alterations in counts of distinct immune cells represent an accessible metric to inform patient care decisions or predict disease outcomes.

T cell phenotypes in COVID-19 - a living review.

COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients' long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation.

Peptide-major histocompatibility complex dimensions control proximal kinase-phosphatase balance during T cell activation.

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

Investigating the Effect of Encapsulation Processing Parameters on the Viability of Therapeutic Viruses in Electrospraying.

The ability of viruses to introduce genetic material into cells can be usefully exploited in a variety of therapies and also vaccination. Encapsulating viruses to limit inactivation by the immune system before reaching the desired target and allowing for controlled release is a promising strategy of delivery. Conventional encapsulation methods, however, can significantly reduce infectivity. The aim of this study was to investigate electrospraying as an alternative encapsulation technique. Two commonly used therapeutic viruses, adenovirus (Ad) and modified vaccinia Ankara (MVA), were selected. First, solutions containing the viruses were electrosprayed in a single needle configuration at increasing voltages to examine the impact of the electric field. Second, the effect of exposing the viruses to pure organic solvents was investigated and compared to that occurring during coaxial electrospraying. Infectivity was determined by measuring the luminescence produced from lysed A549 cells after incubation with treated virus. Neither Ad nor MVA exhibited any significant loss in infectivity when electrosprayed within the range of electrospraying parameters relevant for encapsulation. A significant decrease in infectivity was only observed when MVA was electrosprayed at the highest voltage, 24 kV, and when MVA and Ad were exposed to selected pure organic solvents. Thus, it was concluded that electrospraying would be a viable method for virus encapsulation.

Inflammasome-Mediated Immunogenicity of Clinical and Experimental Vaccine Adjuvants.

In modern vaccines, adjuvants can be sophisticated immunological tools to promote robust and long-lasting protection against prevalent diseases. However, there is an urgent need to improve immunogenicity of vaccines in order to protect mankind from life-threatening diseases such as AIDS, malaria or, most recently, COVID-19. Therefore, it is important to understand the cellular and molecular mechanisms of action of vaccine adjuvants, which generally trigger the innate immune system to enhance signal transition to adaptive immunity, resulting in pathogen-specific protection. Thus, improved understanding of vaccine adjuvant mechanisms may aid in the design of "intelligent" vaccines to provide robust protection from pathogens. Various commonly used clinical adjuvants, such as aluminium salts, saponins or emulsions, have been identified as activators of inflammasomes - multiprotein signalling platforms that drive activation of inflammatory caspases, resulting in secretion of pro-inflammatory cytokines of the IL-1 family. Importantly, these cytokines affect the cellular and humoral arms of adaptive immunity, which indicates that inflammasomes represent a valuable target of vaccine adjuvants. In this review, we highlight the impact of different inflammasomes on vaccine adjuvant-induced immune responses regarding their mechanisms and immunogenicity. In this context, we focus on clinically relevant adjuvants that have been shown to activate the NLRP3 inflammasome and also present various experimental adjuvants that activate the NLRP3-, NLRC4-, AIM2-, pyrin-, or non-canonical inflammasomes and could have the potential to improve future vaccines. Together, we provide a comprehensive overview on vaccine adjuvants that are known, or suggested, to promote immunogenicity through inflammasome-mediated signalling.

Technological Approaches for Improving Vaccination Compliance and Coverage.

Vaccination has been well recognised as a critically important tool in preventing infectious disease, yet incomplete immunisation coverage remains a major obstacle to achieving disease control and eradication. As medical products for global access, vaccines need to be safe, effective and inexpensive. In line with these goals, continuous improvements of vaccine delivery strategies are necessary to achieve the full potential of immunisation. Novel technologies related to vaccine delivery and route of administration, use of advanced adjuvants and controlled antigen release (single-dose immunisation) approaches are expected to contribute to improved coverage and patient compliance. This review discusses the application of micro- and nano-technologies in the alternative routes of vaccine administration (mucosal and cutaneous vaccination), oral vaccine delivery as well as vaccine encapsulation with the aim of controlled antigen release for single-dose vaccination.

Publisher Correction: Adjuvanting a viral vectored vaccine against pre-erythrocytic malaria.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Vaccination with the Staphylococcus aureus secreted proteins EapH1 and EapH2 impacts both S. aureus carriage and invasive disease.

INTRODUCTION: There is a need for an efficacious vaccine reducing infections due to Staphylococcus aureus, a common cause of community and hospital infection. Infecting organisms originate from S. aureus populations colonising the nares and bowel. Antimicrobials are widely used to transiently reduce S. aureus colonisation prior to surgery, a practice which is selecting for resistant S. aureus isolates. S. aureus secretes multiple proteins, including the protease inhibitors extracellular adhesion protein homologue 1 and 2 (EapH1 and EapH2). METHODS: Mice were vaccinated intramuscularly or intranasally with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins, or with control viruses. Using murine S. aureus colonisation models, we monitored S. aureus colonisation by sequential stool sampling. Monitoring of S. aureus invasive disease after intravenous challenge was performed using bacterial load and abscess numbers in the kidney. RESULTS: Intramuscular vaccination with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins significantly reduces bacterial recovery in the murine renal abscess model of infection, but the magnitude of the effect is small. A single intranasal vaccination with an adenoviral vaccine expressing these proteins reduced S. aureus gastrointestinal (GI) tract colonisation. CONCLUSION: Vaccination against EapH1 / EapH2 proteins may offer an antibiotic independent way to reduce S. aureus colonisation, as well as contributing to protection against S. aureus invasive disease.

The HLA A*0201-restricted hTERT(540-548) peptide is not detected on tumor cells by a CTL clone or a high-affinity T-cell receptor.

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I-associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells. Much of this interest has focused on peptide 540-548, ILAKFLHWL, which was predicted to exhibit the strongest binding to the common HLA A*0201 presenting molecule. The hTERT(540-548) peptide is currently being assessed in therapeutic vaccination trials; however, there is controversy surrounding whether it is naturally processed and presented on the surface of neoplastic cells. Here, we generate two highly sensitive reagents to assess the presentation of hTERT(540-548) on tumor cells: (a) a CD8(+) CTL clone, and (b) a recombinant T-cell receptor (TCR) that binds with picomolar affinity and a half-life exceeding 14 h. This TCR enables the identification of individual HLA A2-hTERT(540-548) complexes on the cell surface. The use of both this TCR and the highly antigen-sensitive CTL clone shows that the hTERT(540-548) peptide cannot be detected on the surface of tumor cells, indicating that this peptide is not a naturally presented epitope. We propose that, in future, rigorous methods must be applied for the validation of peptide epitopes used for clinical applications.

Adjuvanting a viral vectored vaccine against pre-erythrocytic malaria.

The majority of routinely given vaccines require two or three immunisations for full protective efficacy. Single dose vaccination has long been considered a key solution to improving the global immunisation coverage. Recent infectious disease outbreaks have further highlighted the need for vaccines that can achieve full efficacy after a single administration. Viral vectors are a potent immunisation platform, benefiting from intrinsic immuno-stimulatory features while retaining excellent safety profile through the use of non-replicating viruses. We investigated the scope for enhancing the protective efficacy of a single dose adenovirus-vectored malaria vaccine in a mouse model of malaria by co-administering it with vaccine adjuvants. Out of 11 adjuvants, only two, Abisco®-100 and CoVaccineHTTM, enhanced vaccine efficacy and sterile protection following malaria challenge. The CoVaccineHTTM adjuvanted vaccine induced significantly higher proportion of antigen specific central memory CD8+ cells, and both adjuvants resulted in increased proportion of CD8+ T cells expressing the CD107a degranulation marker in the absence of IFNγ, TNFα and IL2 production. Our results show that the efficacy of vaccines designed to induce protective T cell responses can be positively modulated with chemical adjuvants and open the possibility of achieving full protection with a single dose immunisation.

Development of persistent gastrointestinal S. aureus carriage in mice.

One fifth to one quarter of the human population is asymptomatically, naturally and persistently colonised by Staphylococcus aureus. Observational human studies indicate that although the whole population is intermittently exposed, some individuals lose S. aureus rapidly. Others become persistent carriers, as assessed by nasal cultures, with many individuals colonised for decades. Current animal models of S. aureus colonisation are expensive and normally require antibiotics. Importantly, these animal models have not yet contributed to our poor understanding of the dichotomy in human colonisation status. Here, we identify a single strain of S. aureus found to be persistently colonising the gastrointestinal tract of BALB/c mice. Phylogenetic analyses suggest it diverged from a human ST15 lineage in the recent past. We show that murine carriage of this organism occurs in the bowel and nares, is acquired early in life, and can persist for months. Importantly, we observe the development of persistent and non-persistent gastrointestinal carriage states in genetically identical mice. We developed a needle- and antibiotic-free model in which we readily induced S. aureus colonisation of the gastrointestinal tract experimentally by environmental exposure. Using our experimental model, impact of adaptive immunity on S. aureus colonisation could be assessed. Vaccine efficacy to eliminate colonisation could also be investigated using this model.

The S. aureus 4-oxalocrotonate tautomerase SAR1376 enhances immune responses when fused to several antigens.

A persistent goal of vaccine development is the enhancement of the immunogenicity of antigens while maintaining safety. One strategy involves alteration of the presentation of the antigen by combining antigens with a multimeric scaffold. Multi-antigen vaccines are under development, and there are presently far more candidate antigens than antigen scaffolding strategies. This is potentially problematic, since prior immunity to a scaffold may inhibit immune responses to the antigen-scaffold combination. In this study, a series of domains from S. aureus which have been shown to crystallise into multimeric structures have been examined for their scaffolding potential. Of these domains, SAR1376, a 62 amino acid member of the 4-oxalocrotonate tautomerase (4-OT) family, was pro-immunogenic in mice when fused to a range of pathogen antigens from both S. aureus and P. falciparum, and delivered by either DNA vaccination, viral vector vaccines or as protein-in-adjuvant formulations. The adjuvant effect did not depend on enzymatic activity, but was abrogated by mutations disrupting the hexameric structure of the protein. We therefore propose that SAR1376, and perhaps other members of the 4-OT protein family, represent very small domains which can be fused to a wide range of antigens, enhancing immune responses against them.