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Background: Seasonal influenza vaccination with a standard trivalent influenza vaccine (TIV) induces a modest, and cross-reactive, Fc functional antibody response in older adults. Recent improvements to influenza vaccines include a quadrivalent influenza vaccine (QIV) and a TIV adjuvanted with the squalene-based oil-in-water emulsion MF59. Methods: Pre- and post-vaccination serum samples from older adults vaccinated with QJV (n = 27) and adjuvanted TIV (n = 44) were studied using hemagglutination inhibition (HAI) assays and dimeric Fc-gamma receptor IIIa binding ELISAs, as a surrogate of antibody-dependent cellular cytotoxicity (ADCC). Results: We found that the unadjuvanted QIV elicited a stronger HAI response against the H1N1 vaccine virus than the adjuvanted TIV. Post-vaccination levels of HA-specific ADCC antibodies were similar for older adults vaccinated with QIV and adjuvanted TIV. The ADCC response to influenza vaccination was largely determined by pre-vaccination or baseline levels of these antibodies, with older adults with low baseline levels of ADCC activity demonstrating greater post-vaccination rises. Conclusions: In this cohort of community-dwelling older adults, the QIV was at least as good as the adjuvanted TIV in the induction of ADCC and HAI responses. Further studies on how these antibody responses translate to efficacy in preventing influenza infections are warranted.

Globally the most commonly utilised immunisation against influenza is the trivalent inactivated influenza vaccine (TIV) derived from an A/H1N1, an A/H3N2 and aB type influenza virus. Vaccine effectiveness of TIV varies year to year, depending on how well antigenically matched the strains in the vaccine are compared to circulating strains [1,2]. Moreover, vaccine effectiveness can vary within certain subpopulations such as HIV-positive, young children and the elderly. Decreased vaccine effectiveness in the elderly is associated with impaired Ab production, as measured by standard hemagglutination inhibition (HAI) assays. We investigated the level of Antibody Dependent Phagocytosis (ADP)-mediating Abs induced by the 2008-TIV in healthy Australian adults aged over and under 60years to determine if this immune function was also reduced in the elderly. We utilised an ADP assay that measures the uptake of IgG-opsonised HA-coated fluorescent microspheres by a monocytic cell line. We also measured HA-specific Abs that are close enough to bind to dimeric FcγRIIa ectodomains in an ELISA-based assay. Furthermore, we compared the extent of cross-reactive recognition of diverse influenza strains by ADP-mediating Abs found in pre- and post-vaccination sera in both of these groups. We found that young adults and older adults mounted similar ADP activity against HAs contained in the 2008-TIV, despite older adults have diminished HI responses. The level of cross-reactive antibodies against other HAs was limited in both groups. We conclude that seasonal influenza vaccination elicits limited cross-reactive ADP to HA in both young and older adults. New influenza vaccination strategies that elicit cross-reactive and polyfunctional antibodies are needed.

A safe and effective vaccine against HIV is a global health priority. Large-scale phase III clinical vaccine trials based on neutralizing antibodies and cytotoxic T-lymphocytes have failed to provide protection, highlighting the lack of understanding of basic immune correlates of protection against HIV. The partial success of the RV144 vaccine trial, however, sparked an intense research effort to identify and describe the protective potential of non-neutralizing antibodies. Correlates of protection analyses have identified antibodies that induced antibody-dependent cellular cytotoxicity (ADCC) as potentially important. Despite the attractiveness of utilizing ADCC antibodies for HIV vaccine design, it is important to note that effective ADCC responses are contingent on many factors. As discussed in this review, these factors are important considerations for determining the feasibility of designing an optimal ADCC antibody-inducing vaccine construct. Important determinants of ADCC responses include characteristics of the antibody, such as isotype and subclass, antigen-specificity, titer, durability and glycosylation of the constant region. Second, ADCC immune responses are highly contingent on the natural killer (NK) cell effectors. This review will describe the current state of knowledge regarding the ontogeny of NK cells, highlighting the continuous “education” they undergo that determines their functional potential upon stimulation. Other important NK cell factors, such as constant region receptor polymorphisms, cellular exhaustion, and the effects of the cytokine milieu on cellular function, will also be covered. Finally, an exciting, but yet untested, role for NK cell-mediated ADCC lies in its potential ability to eliminate latently infected cells, which harbor the viral reservoir. The review will address the potential of a two-pronged attack, where latently infected cells are induced to express HIV antigens and then eliminated by NK cells via an ADCC mechanism, with the goal of inducing a cure.

Background Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. Methods We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. Results A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. Conclusions We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV.

Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.

DNA prime and recombinant fowlpox virus (rFPV) boost vaccines were designed to express multiple HIV or SIV antigens for use in human clinical trials and in pre-clinical trials in macaques. Three sets of vaccines with matching HIV or SIV antigen sets, modified for vaccine safety considerations, were constructed and shown to express the relevant proteins. The rFPV vaccines with inserts at up to three sites, were stable on passage in chick cell culture, including during GMP manufacture of vaccines for human Phase I clinical trials. Cellular and humoral immunogenicity in mice was demonstrated using a DNA prime/rFPV boost and vaccinia virus challenge model. These data establish a preliminary safety and efficacy profile for these multigenic vaccines suggesting they are suitable for advanced development as candidate HIV vaccines.

To induce broad T cell immunity to HIV-1, we evaluated the safety, immunogenicity and dose-response relationship of DNA and recombinant Fowlpoxvirus (rFPV) vaccines encoding five shared HIV subtype AE genes (Gag, Pol, Env, Tat, Rev) in pigtail macaques. The DNA (three doses of either 1 mg or 4.5 mg) and rFPV (a single boost of either 5 x 10(7) or 2 x 10(8) plaque forming units) vaccines were administered intramuscularly without adjuvants. Broadly reactive HIV-specific T cell immunity was stimulated by all doses of the vaccines administered, without significant differences between the high and low doses studied. The vaccines induced both CD4 and CD8 T cell responses to Gag, Pol, Env and Tat/Rev proteins, with CD4 T cell responses being greater in magnitude than CD8 T cell responses. The vaccine-induced T cell responses had significant cross-recognition of heterologous HIV-1 proteins from non-AE HIV-1 subtypes. In conclusion, these subtype AE HIV-1 DNA and rFPV vaccines were safe, induced broad T-cell immunity in macaques, and are suitable for progression into clinical trials.

We developed highly expressing clade B and AE DNA and envelope protein (Env) vaccines for evaluation in mice and macaques as DNA prime/protein boost regimens. High levels of Env-specific antibodies were induced in mice, albeit with limited neutralizing activity in vitro. A combined clade B and AE regimen induced high titer Env-specific antibody in two pigtail macaques that neutralized several strains of HIV-1. However, upon mucosal challenge with SHIV(SF162P3) no protection from infection was observed. Although the vaccines tested provide a platform for inducing robust humoral immunity, further refinements to broaden coverage against divergent strains and induce mucosal immunity are needed.

Induction of high levels of broadly reactive cytotoxic T lymphocytes (CTL) remains a promising approach for an effective HIV-1 vaccine. We have developed a novel genetic-based vaccine strategy that encodes consensus overlapping peptide sets from all HIV-1 proteins scrambled together. This synthetic scrambled antigen vaccine (SAVINE) strategy has significant advantages, e.g. capacity to encode more antigens safely and is very flexible compared to traditional isolate-based strategies. The SAVINE vaccine strategy is clearly immunogenic, being able to restimulate a range of human HIV-1 specific responses in vitro and induce HIV-1 specific immunity in vivo in mice. Interestingly, different in vivo delivery strategies affected the resulting immunity and immunodominance pattern in mice. This platform strategy could be used for other infections and cancers where T cell responses are important for protection.

Further advances are required in understanding protection from AIDS by T cell immunity across mucosal sites of virus transmission. We analysed a set of multigenic HIV and SHIV DNA and Fowlpoxvirus (FPV) prime and boost vaccines for immunogenicity and protective efficacy in outbred pigtail macaques when delivered via mucosal surfaces (intranasally or intrarectally). Intranasally delivered DNA, even when adjuvanted and given as a fine droplet spray, was neither immunogenic nor protective in macaques. Some protection from acute infection with a pathogenic vaginal SHIVSF162P3 challenge was, however, observed with a regimen involving intramuscular DNA vaccine priming followed by either intranasally or intrarectally delivered rFPV boosting. Interestingly, animals boosted with rFPV vaccine via either of these mucosal routes had poor circulating T cell responses prior to challenge with SHIV compared to those boosted via the intramuscular route. Nevertheless, the mucosally-vaccinated animals generated equivalent anamnestic mucosal and systemic SHIV-specific CD4 and CD8 T cell responses following SHIV administration, with significant reduction in acute plasma viremia against this vaginal challenge. Our data suggest strategies for effective priming of partial immunity to mucosal HIV-1 exposure utilizing systemic prime and mucosal boost vaccination strategies.

Induction of HIV-specific T-cell responses by vaccines may facilitate efficient control of HIV. Plasmid DNA vaccines and recombinant fowlpoxvirus (rFPV) vaccines are promising HIV-1 vaccine candidates, although either vaccine alone may be insufficient to protect against HIV-1. A consecutive immunisation strategy involving priming with DNA and boosting with rFPV vaccines encoding multiple common HIV-1 antigens was further evaluated in 30 macaques. The DNA vaccine vector included CpG immunostimulatory molecules, and rFPV vaccines were compared with rFPV vaccines co-expressing the pro-T cell cytokines IFNgamma or IL-12. Vaccines expressed multiple HIV-1 genes, mutated to remove active sites of the HIV proteins. The vaccines were well tolerated, and a significant enhancement of DNA-vaccine primed HIV-1 specific T lymphocyte responses was observed following rFPV boosting. Co-expression of IFNgamma or IL-12 by the rFPV vaccines did not further enhance immune responses. Non-sterilising protection from a non-pathogenic HIV-1 challenge was observed. This study provides evidence of a safe, optimised, strategy for the generation of T-cell mediated immunity to HIV-1.

Complex recombinant fowlpoxvirus (rFPV) vaccines expressing both HIV-1 antigens and type 1 cytokines could facilitate the induction of cellular immunity against HIV-1. A single rFPV expressing both HIV-1gag/pol and human interferon-g (FPVgag/ pol-IFNg) was constructed and assessed as a therapeutic vaccine for safety and immunogenicity in macaques (Macaca nemestrina) previously infected with HIV-1. FPV gag/pol-IFNg vaccinations were safe and enhanced T cell proliferative responses to Gag antigens (but not control tetanus antigens). Enhanced CTL responses to gag/pol antigens were also observed following IFNg expressing vaccinations. Since cellular immunity may be critical to controlling or preventing HIV-1 infection, these observations suggest that avipox vectors co-expressing IFNg should be further evaluated as therapeutic or preventive HIV1 vaccines. 7 2000 Elsevier Science Ltd. All rights reserved.