The capacity of systemic rDNA/virus vector heterologous prime-boost immunisation to elicit strong, protective T cell-mediated immune responses against HIV or SIV challenge in non-human primates has been extensively studied [11
]. However, following disappointing outcomes of HIV-1 rDNA/virus prime-boost phase I clinical trials, there is now a great push to improve the immunogenicity of DNA vaccines in humans and also to develop vaccines that generate good mucosal immunity, since mucosae are the primary sites of HIV infection. Here, we have performed a comprehensive study of rDNA and viral vector vaccine administration and evaluated the capacity of combined mucosal/systemic delivery routes to enhance both mucosal and systemic humoral and cell-mediated immunity to encoded HIV-1 vaccine antigens. In the second part of the study, we have mainly compared the best combined rDNA viral prime-boost immunisation strategy (rDNA/rFPV) to the best pox viral prime-boost immunisation strategy (rFPV/rVV) and evaluated both their protective efficacy and immunogenicity against an immunodominant Kd
epitope, including the avidity of vaccine-induced CD8+ T cells.
Delivery of rDNA vaccines i.n., either with or without lipid complexes, or i.m via needle delivery, has largely been ineffective at generating good mucosal immunity to encoded vaccine antigens [13
], except after i.n. co-adminstration of cholera toxin has albeit with serious side effects [37
]. We have previously tested i.n. delivery of lipid-complexed DNA followed by i.n. boosting with FPV HIV constructs and have found that this approach generated sub-optimal T cell immune responses in both mice and non-human primates (Ranasinghe and Ramsay unpublished observations) [24
]. In the present study we compared different prime-boost immunisation strategies (i.n. 2× DNA-HIV/i.m. 2× FPV-HIV vs i.m. 2× DNA-HIV/i.n. 2× FPV-HIV) and have found that mucosal delivery of rFPV was greatly superior at generating strong sustained mucosal immune responses compared to i.n. DNA delivery. These data indicate that rFPV is an excellent and safe mucosal delivery vector similar to rMVA [39
] or the poxvirus vector tiantan vaccinia (TiVV) [42
] or NYVAC [43
]. Our phase I human clinical trials have also clearly demonstrated that systemic delivery of rFPV was extremely safe in humans [1
], although the effects of rFPV following mucosal delivery have not yet been clinically tested in humans. Current observations may be important for the development of clinical trials of poxvirus vector-based mucosal HIV-1 vaccines in the future.
In the current study, strong sustained memory mucosal T cell responses were observed in mucosal immune compartment (genito-rectal nodes, vaginal tissues and lung tissues), as well as in the systemic immune compartment (spleen) following i.m. 2× DNA-HIV/i.n. 2× FPV-HIV immunisation. These findings are also consistent with those of a recent i.n. TiVV-HIV-1 Gag prime/i.m. rDNA boost immunisation study demonstrating enhanced Gag-specific mucosal and systemic T and B cell immunity following i.m. rDNA delivery [42
]. Surprisingly, only low-level mucosal T cell responses were observed following i.m. 2× DNA-HIV/i.r. 2× FPV-HIV immunisation, this could possibly be due to poor i.r. uptake of rFPV in mice unlike in macaques [24
]. On the contrary, following i.m. 2×DNA-HIV/i.m. 2×FPV-HIV immunisation even though lower IFN-γSFU was observed in genito-rectal nodes, similar levels of IFN-γ SFU to i.m. 2×DNA-HIV/i.n. 2× FPV-HIV delivery was recorded in vaginal tissues, lung tissues and spleen. Few studies have shown that purely systemic vaccination can induce mucosal responses, but whether these responses are effective or sustained long term has been highly debated [44
]. These studies further substantiate that the route of vaccine delivery can significantly influence the magnitude, immunodominance hierarchy and/or duration of resultant antibody and CTL responses [25
Furthermore, the T cell depletion studies also demonstrated the influence of route of vaccine delivery on immunity. Interestingly, following i.m. 2× DNA-HIV/i.n. 2× FPV-HIV prime-boost immunisation enhanced systemic memory CD8+ T cell responses as well as CD4+ T cell responses, were detected compared to purely systemic (i.m./i.m.) or i.m./i.r immunisation strategies. The enhanced CD4+ T cell responses following i.m./i.n. delivery may also substantiate the elevated serum p24 Gag-specific IgG1 and IgG2a antibody levels that were observed following this vaccination. Surprisingly, low IgG2a serum antibody levels were observed following purely systemic routes of immunisation, and out of the three DNA immunisation strategies tested, only the i.m./i.n. combination generated measurable mucosal antibody responses (IgG and IgA) to p24 Gag. The reason why only the second i.n. rFPV booster immunisation (not the second i.r. or i.m. rFPV), enhanced the magnitude of antibody response, warrants further investigation. It is noteworthy that in previous studies, we have also found that single i.n. FPV-HIV/i.m. VV-HIV (poxvirus/poxvirus) prime-boost immunisation did not generate good p24 Gag-specific IgGantibody responses[25
]. These current observations further indicate that the route of vaccine delivery and/or number of booster immunisations received can be critical factors when evaluating novel vector vaccine strategies for HIV-1.
Recently, we have shown that avidity of HIV-specific CD8+ T cells can also be modulated by the route of vaccine delivery [26
]. Even though avidity of vaccine-induced T cell responses is an important factor in evaluating protective immunity [47
] it is often overlooked when evaluating vaccine responses, possibly due to difficulties associated with assay techniques. Comparison of i.m. 2× DNA-HIV/i.n. FPV HIV delivery with poxvirus prime-boost immunisation, clearly showed that the latter approach generated enhanced systemic T cell immunity as measured by IFN-γ ELISpot and tetramer staining. The combined mucosal (i.n.) and systemic (i.m.) FPV-HIV/VV-HIV immunisation generated the most robust systemic and mucosal T cell responses against the Kd
epitope. Furthermore, when the avidities of vaccine-induced CD8+ T cells were compared following i.m. 2× DNA-HIV/i.n. FPV-HIV and i.n. FPV-HIV/i.m. VV-HIV immunisation strategies it was found that, the poxvirus/poxvirus prime-boost immunisation regime generated CTL of heightened avidity correlating with better protection against influenza virus- Kd
mucosal challenge. In an elegant study, Belyakov et al. have also shown that a mucosal peptide prime/poxvirus boost immunisation can induce greater number of high avidity mucosal CD8+ T cells that can control systemic dissemination of i.r. administered pathogenic SHIV in rhesus macaques and this protection correlated better with induction of mucosal CD8+ T cells than systemic CD8+ T cells [48
]. These results are highly consistent with our earlier comparisons of T cell avidities resulting from combined mucosal/systemic poxvirus immunisation strategies, with purely systemic immunisation regimes, eliciting that the latter immunisation strategy generated CTL of lower avidity as measured by tetramer dissociation [26
], or lower protection following influenza virus- Kd
mucosal challenge (Ranasinghe unpublished observations), similar to that observed here following i.m. 2× DNA-HIV/i.n. FPV-HIV immunisation. Interestingly, the magnitude of mucosal responses generated by this strategy was very similar to that of pure systemic (i.m./i.m.) FPV-HIV/VV-HIV delivery, although levels of systemic CD8+ T cell responses were greater in the latter case. These observations confirm that the magnitude of T cell responses as measured by IFN-γ production does not always correlate with T cell avidity or with protective efficacy [26
], serving to underline a major caveat in the interpretation of vaccine studies.
Following combined mucosal/systemic prime-boost immunisation, the majority of Gag-specific IFN-γ producing CD8+ T cells were found to be CD107a-positive, suggestive of high levels of cytolytic activity. Furthermore, increased levels of CD62L expression indicated that a greater percentage of central memory CTL were generated following poxvirus prime-boosting compared to i.m. 2× DNA-HIV/i.n. FPV-HIV immunisation. More significant weight loss after challenge with influenza-Kd
virus was observed in mice vaccinated via rDNA/rFPV prime-boost compared to the i.n./i.m. poxvirus/poxvirus regime, especially at day 5 and 6 post-challenge (p
= 0.0256 and p
= 0.0287 respectively), although mice in both groups recovered by day 10. It is noteworthy that even the unimmunised mice, that lost up to 20–25% of their body weight, showed significant weight gain by day 9 post-challenge, a common finding in this influenza challenge model. Interestingly, CTL avidity curves ( and ) were also consistent with the protection data. Evaluation of T cell avidities at 5–6 days following influenza-Kd
virus challenge may reveal greater differences between the two vaccine groups and further studies have been designed to clarify this point. Previous studies have established that (i) high avidity CD8+ T cells are generated during early stages of pathogen infection but subsequently low-avidity CD8+ T cells can persist in chronic infections, and (ii) also T cell populations can be expanded following infection and that avidity modulation, either through infection or immunisation, may play an important role in the nature of the CD8+ T cell responses that are generated in vivo
Antigen-specific polyfunctional CD4+ or CD8+ T cells that express IFN-γ, IL-2 and TNF-α are thought to be a hallmark of protective immunity [50
]. Indeed, there are distinct differences in the potency of effector cells based on their polyfunctional cytokine secretion profiles [53
]. However, the association of high avidity T cells with polyfunctional cytokine effector function is still unclear [54
], although our studies indicate that these two functional activities may be closely related. Mice that received i.m. 2×DNA-HIV/i.n. FPV-HIV immunisation had reduced numbers of IFN-γ, IL-2 and TNF-α producing CD8+ T cells and also a reduction in total numbers of CD8+ T cells post challenge. Interestingly, antibody arrays also indicated that unlike rDNA/poxvirus prime-boost immunisation, the poxvirus/poxvirus prime-boost immunisation generated effector CD8+ T cells that expressed a wide range of cytokines and chemokines (IFN-γ, IL-3, IL-6, GM-CSF, CCL3, CCL5, and CCL9) 16 h following peptide stimulation. Our recent findings indicate that if profiles were measured at an earlier time point (4–5 h) a much broader cytokine profile would have been observed (i.e. IL-2 and TNF-α), as expression kinetics of different cytokines/chemokines are highly time dependent (Ranasinghe unpublished observations).
In conclusion, following rDNA vaccination in order to generate heightened mucosal T cell immunity and antibody responses two consecutive FPV-HIV booster immunisations were required. Out of the rDNA delivery strategies tested, i.m. 2× DNA-HIV/i.n. 2× FPV-HIV prime-boost immunisation generated the best mucosal and systemic cell-mediated and antibody responses to encoded vaccine antigens. However, the poxvirus/poxvirus prime-boost strategy (i.n. FPV-HIV/i.m. VV-HIV) elicited more robust and sustained effector/memory mucosal and systemic CD8+ T cell responses, with enhanced cytokine/chemokine profiles, T cell avidity and protective efficacy compared to the heterologous i.m. 2× DNA-HIV/i.n. FPV-HIV prime-boost immunisation. Hence, we believe that combined mucosal/systemic prime-boost immunisation strategies have considerable potential for the further development of HIV-1 vaccines.