Heterologous Two-Dose Vaccination with Simian Adenovirus and Poxvirus Vectors Elicits Long-Lasting Cellular Immunity to Influenza Virus A in Healthy Adults.

BACKGROUND: T-cell responses against highly conserved influenza antigens have been previously associated with protection. However, these immune responses are poorly maintained following recovery from influenza infection and are not boosted by inactivated influenza vaccines. We have previously demonstrated the safety and immunogenicity of two viral vectored vaccines, modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAdOx1 expressing conserved influenza virus antigens, nucleoprotein (NP) and matrix protein-1 (M1). We now report on the safety and long-term immunogenicity of multiple combination regimes of these vaccines in young and older adults. METHODS: We conducted a Phase I open-label, randomized, multi-center study in 49 subjects aged 18-46years and 24 subjects aged 50years or over. Following vaccination, adverse events were recorded and the kinetics of the T cell response determined at multiple time points for up to 18months. FINDINGS: Both vaccines were well tolerated. A two dose heterologous vaccination regimen significantly increased the magnitude of pre-existing T-cell responses to NP and M1 after both doses in young and older adults. The fold-increase and peak immune responses after a single MVA-NP+M1 vaccination was significantly higher compared to ChAdOx1 NP+M1. In a mixed regression model, T-cell responses over 18months were significantly higher following the two dose vaccination regimen of MVA/ChAdOx1 NP+M1. INTERPRETATION: A two dose heterologous vaccination regimen of MVA/ChAdOx1 NP+M1 was safe and immunogenic in young and older adults, offering a promising vaccination strategy for inducing long-term broadly cross-reactive protection against influenza A. FUNDING SOURCE: Medical Research Council UK, NIHR BMRC Oxford.

Effect of Loxosceles gaucho venom on cell morphology and behaviour in vitro in the presence and absence of sphingomyelin.

This study was performed to investigate whether the toxic effects of Loxosceles gaucho venom on cells might be exerted via stimulators of TNF-alpha release generated by sphingomyelinase D--a major component of the venom. It was demonstrated that L. gaucho venom alone is unable to induce TNF-alpha release by J774A.1 cells, while in the presence of exogenous sphingomyelin it induces a high level of TNF-alpha release which is significantly increased by incubation with non-inactivated serum. Ceramide phosphate also induces TNF-alpha release in J774A.1 cells, but (unlike sphingomyelin/sphingomyelinase) the level of release is not influenced by the presence or otherwise of non-inactivated serum. L. gaucho venom does not induce proliferation of J774A.1 cells and even at high concentrations it does not affect their viability. J774A.1 cells, which prior to venom treatment were elongated and clumped, round up after venom treatment, but, revert to their original morphology after incubation with fresh medium. TNF-alpha resistant MRC-5 cells and TNF-alpha sensitive MCF-7 cells are susceptible to the toxic effect of both L. gaucho venom and ceramide phosphate. The results obtained in this study demonstrate that exogenous sphingomyelin can modulate, in vitro, the release of TNF-alpha induced by L. gaucho venom in mouse macrophages. In addition, the results also indicate that ceramide phosphate and L. gaucho venom are toxic to several different cell types, via a variety of mechanisms, some, but not all, of which may involve TNF-alpha as an intermediary.

Influence of sphingomyelin and TNF-alpha release on lethality and local inflammatory reaction induced by Loxosceles gaucho spider venom in mice.

It is well known that Loxosceles venom induces local dermonecrosis in rabbits, guinea pigs and humans but not in mice, although, depending on the dose, Loxosceles venom can be lethal to mice. In this work we demonstrate that mice injected intradermally in the dorsal area of the back can survive a lethal dose of Loxosceles gaucho venom and also develop an inflammatory reaction (with infiltration of leukocytes shown by histological analysis) at the local injection site when the venom is co-administered with sphingomyelin. It was observed that more venom was retained for a longer period of time at the local injection site when venom was co-administered with sphingomyelin. The presence of exogenous sphingomyelin did not influence significantly the release of TNF-alpha induced by L. gaucho venom. These results suggest that the action of venom on sphingomyelin, producing ceramide phosphate, causes the development of an inflammatory reaction, which in turn traps the venom in the local area for a long period of time and does not allow it to disperse systemically in a dose sufficient to cause death. Our findings also indicate that the size and availability of the local sphingomyelin pool may be important in determining the outcome of Loxosceles envenoming in different mammalian species.

Ability of SPI2 mutant of S. typhi to effectively induce antibody responses to the mucosal antigen enterotoxigenic E. coli heat labile toxin B subunit after oral delivery to humans.

We have evaluated an oral vaccine based on an Salmonella enteric serovar typhi (S. typhi) Ty2 derivative TSB7 harboring deletion mutations in ssaV (SPI-2) and aroC together with a chromosomally integrated copy of eltB encoding the B subunit of enterotoxigenic Escherichia coli heat labile toxin (LT-B) in volunteers. Two oral doses of 10(8) or 10(9)CFU were administered to two groups of volunteers and both doses were well tolerated, with no vaccinemia, and only transient stool shedding. Immune responses to LT-B and S. typhi lipopolysaccharide were demonstrated in 67 and 97% of subjects, respectively, without evidence of anti-carrier immunity preventing boosting of LT-B responses in many cases. Further development of this salmonella-based (spi-VEC) system for oral delivery of heterologous antigens appears warranted.

Mucosal and systemic immune responses to plasmid protein pgp3 in patients with genital and ocular Chlamydia trachomatis infection.

The circulating and cervical B cell responses to Chlamydia trachomatis plasmid protein pgp3 were characterized in children and adults with ocular or genital chlamydial infection using the enzyme-linked immunospot assay (ELISPOT) and ELISA. No pgp3-specific ASCs were detected in healthy controls, but predominantly IgA ASCs were detected in UK adults with uncomplicated cervicitis or urethritis (P = 0.03, 0.019). In patients with extragenital complications or pelvic inflammatory disease a mixed response with more IgG and IgM ASCs was evident, suggesting a breach of mucosal immune compartmentalization with more extensive infection. In women with chlamydial cervicitis, ASCs secreting predominantly IgA, but also IgG, to pgp3 were present in cervix at presentation, with a frequency 30-50 times higher than blood. Cervical ASC numbers, especially IgG, fell markedly six weeks after antibiotic treatment. We detected principally IgA pgp3-specific antibody secreting cells (ASCs) in children resident in a Gambian endemic area, with a trend towards suppression of IgA responses during intense trachomatous inflammation (P = 0.06), as previously reported for other chlamydial antigens, and in keeping with the findings in genital disease. These data provide a rationale for further studies of immune responses to pgp3 in humans and animal models of chlamydia-induced disease, and its potential use in diagnostic assays and protective immunization strategies.

Gastrointestinal immune responses in HIV infected subjects

The gut associated lymphoid tissue is responsible for specific responses to intestinal antigens. During HIV infection, mucosal immune deficiency may account for the gastrointestinal infections. In this review we describe the humoral and cellular mucosal immune responses in normal and HIV-infected subjects.