Georgia Deliyannis

ISCOMTM-based vaccines: The second decade

The immunostimulating complex or ‘iscom’ was first described 20 years ago as an antigen delivery system with powerful immunostimulating activity. Iscoms are cage‐like structures, typically 40 nm in diameter, that are comprised of antigen, cholesterol, phospholipid and saponin. ISCOMTM‐based vaccines have been shown to promote both antibody and cellular immune responses in a variety of experimental animal models. This review focuses on the evaluation of ISCOMTM‐based vaccines in animals over the past 10 years, as well as examining the progress that has been achieved in the development of human vaccines based on ISCOMTM adjuvant technology.

Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in mice

BackgroundCigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.MethodsBALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.ResultsInflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.ConclusionSmoke induced inflammation does not protect against influenza infection.In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.

Induction of Long-Term Memory CD8+ T Cells for Recall of Viral Clearing Responses against Influenza Virus

ABSTRACT Induction of cytotoxic T-cell-mediated virus-clearing responses by influenza virus T cell determinant-containing peptide immunogens was examined. The most potent synthetic immunogens for eliciting pulmonary viral-clearing responses contained peptides representing determinants for CD4 and CD8 T cells (TH and CTL peptides, respectively) together with two or four palmitic acid (Pal) groups. Inoculated in adjuvant, these Pal2- or Pal4-CTL-TH lipopeptides and the nonlipidated CTL peptide induced equivalent levels of cytolytic activity in the primary effector phase of the response. The ability to recall lytic responses, however, diminished much more rapidly in CTL peptide-primed than in lipopeptide-primed mice. By 15 months postpriming, the recalled lytic activity in lipopeptide-inoculated mice remained potent, but the response induced by the CTL peptide was weak. Enumeration of specific gamma interferon-secreting CD8 T cells revealed that a greater number of these T cells had entered or remained in the memory pool in lipopeptide-primed mice, arguing for a quantitative rather than qualitative enhancement of the response on recall. Addition of either the lipid or the TH peptide to the CTL peptide was not sufficient to provide these long-lived antiviral responses, but inclusion of both components augmented the response. CD4 T cells elicited by the lipopeptides did not influence the rate of viral clearance upon challenge and most likely had a role in induction or maintenance of the memory response. It therefore appears that the lipopeptide immunogens, although not significantly superior at inducing primary effector CD8 T cells, elicit a much more effective memory population, the recall of which may account for their superiority in inducing pulmonary protection after viral challenge.

Intranasal lipopeptide primes lung-resident memory CD8+ T cells for long-term pulmonary protection against influenza

The longevity of the influenza virus‐specific CD8+ T cell response following intranasal delivery of a synthetic lipopeptide was investigated and the characteristics and location of the cells associated with viral clearance examined. The lipopeptide, incorporating an epitope for CD8+ T cells and another for CD4+ T cells with the lipid moiety S‐[2,3‐bis(palmitoyloxy)propyl]cysteine (Pam2Cys) attached, induced potent and long‐lived pulmonary protection. Both the lipopeptide and its largely unprotective non‐lipidated counterpart elicited comparable numbers of CD8+ T cells in the spleen, which was the main location of the memory pool. However, the lipopeptide, unlike the non‐lipidated peptide, also induced a substantial memory population that remained in the lungs and was rapidly activated upon viral challenge months later. These lipopeptide‐induced lung‐resident CD8+ T cells were also very similar in number and IFN‐γ‐secreting potential to those induced by prior exposure to the virus itself and are likely mediators of initial viral clearance prior to recruitment from the expanding lymph node T cell pool. Significant clearing responses were demonstrated as late as 9 months post‐lipopeptide vaccination. This study shows that CD8+ T cells primed by the lipopeptide are not only long‐lived but can take up residence in the lung where they are important early mediators of pulmonary protection.

Single dose intranasal immunization with ISCOMATRIX vaccines to elicit antibody-mediated clearance of influenza virus requires delivery to the lower respiratory tract.

The effectiveness of single dose, intranasally delivered vaccines comprising detergent-disrupted inactivated influenza virus (split virus) and ISCOMATRIX adjuvant was examined in mice. Vaccines formulated with adjuvant required 10- to 100-fold less split virus antigen to induce pulmonary protection following viral challenge when compared to vaccines containing split virus alone. Furthermore, those formulated with ISCOMATRIX adjuvant elicited specific antibody in serum, saliva, vaginal, nasal and lung fluids when delivered to the entire respiratory tract. No specific antibody was detected in serum or mucosal samples, however, when the same vaccines were delivered using a procedure that restricted the inoculum to the nasal passages. Good protective responses can thus be achieved with only a single intranasal inoculation of influenza vaccine formulated with adjuvant, providing the vaccine can access sites of immune induction in the lower respiratory tract.

Immunopotentiation of humoral and cellular responses to inactivated influenza vaccines by two different adjuvants with potential for human use

Two quite different adjuvants, currently under development for use in humans, have been examined for their effects on the magnitude and type of immunity elicited in response to inactivated influenza vaccine. Immunostimulating complexes (ISCOM adjuvant) contain the saponin ISCOPREP 703, and SPT is an oil-in-water emulsion of squalane, non-ionic block copolymer (L121) and Tween 80. Influenza virus vaccines formulated in either adjuvant were far superior to the non-adjuvanted aqueous vaccine in eliciting antibody and T-cell responses in mice, particularly at lower doses of antigen. In addition, the vaccines containing adjuvant were superior in eliciting protective immunity. One of the shortcomings of the unadjuvanted inactivated influenza vaccine was its inability to elicit a primary proliferative T-cell response. However, after one dose of either adjuvanted vaccine, strong proliferative responses were achieved. We also show that subcutaneous vaccination with inactivated vaccines is capable of modulating the isotype profile of antibody secreting cells generated in the lungs of mice in response to intranasal challenge with live virus. In this system, the isotype of antibody elicited after challenge of mice that had received ISCOM vaccine more closely mimicked that of animals vaccinated with live virus.

The design and proof of concept for a CD8(+) T cell-based vaccine inducing cross-subtype protection against influenza A virus.

In this study, we examined the reactivity of human peripheral blood mononuclear cells to a panel of influenza A virus (IAV) CD8+ T‐cell epitopes that are recognised by the major human leukocyte antigen (HLA) groups represented in the human population. We examined the level of recognition in a sample of the human population and the potential coverage that could be achieved if these were incorporated into a T‐cell epitope‐based vaccine. We then designed a candidate influenza vaccine that incorporated three of the examined HLA‐A2‐restricted influenza epitopes into Pam2Cys‐based lipopeptides. These lipopeptides do not require the addition of an adjuvant and can be delivered directly to the respiratory mucosa enabling the generation of local memory cell populations that are crucial for clearance of influenza. Intranasal administration of a mixture of three lipopeptides to HLA‐A2 transgenic HHD mice elicited multiple CD8+ T‐cell specificities in the spleen and lung that closely mimicked the response generated following natural infection with influenza. These CD8+ T cells were associated with viral reduction following H3N1 influenza virus challenge for as long as 3 months after lipopeptide administration. In addition, lipopeptides containing IAV‐targeting epitopes conferred substantial benefit against death following infection with a virulent H1N1 strain. Because CD8+ T cell epitopes are often derived from highly conserved regions of influenza viruses, such vaccines need not be reformulated annually and unlike current antibody‐inducing vaccines could provide cross‐protective immunity against newly emerging pandemic viruses.

A 320-Kilobase Artificial Chromosome Encoding the Human HLA DR3-DQ2 MHC Haplotype Confers HLA Restriction in Transgenic Mice

MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact ∼320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (Aβnull/null) and expressing only murine CD4. A broad range of TCR Vβ families was used in the peripheral T cell repertoire, which was also purged of Vβ5-, Vβ11-, and Vβ12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the Aβnull/null background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR- and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.

Effect of pre-existing cytotoxic T lymphocytes on therapeutic vaccines

Therapeutic vaccines which aim to induce CD8+ cytotoxic T lymphocyte (CTL) responses will often be required to perform in the presence of pre‐existing CTL which recognize epitopes within the vaccine. Here we explore the ability of a viral vaccine vector presenting several co‐dominant CTL epitopes to prime CTL responses in animals that have a pre‐existing CTL response to one of the epitopes in the vaccine. The vaccine was usually capable of inducing multiple new responses, suggesting that immunodomination effects of pre‐existing CTL may generally be minimal following vaccination. However, when large numbers of pre‐existing CTL were present, a novel type of immune modulation was observed whereby (1) the vaccine failed to prime efficiently new CTL responses that were restricted by the same MHC gene as the pre‐existing responses, and (2) vaccine‐induced CTL responses restricted by other MHC genes were enhanced. These results may have implications for therapeutic multi‐epitope vaccines for diseases like HIV and melanoma, which aim to broaden CTL responses.

Targeting subcapsular antigens for prevention of Klebsiella pneumoniae infections.

Vaccination strategies against Klebsiella pneumoniae have largely focussed on the polysaccharide capsule. However, the large number and high prevalence of individual capsular serotypes limits the widespread applicability of capsule-based vaccines. This study establishes that immunization with purified LPS can protect mice against lethal challenge with K. pneumoniae, and that subcapsular antibodies directed against purified LPS can be used to treat and/or prevent experimental K. pneumoniae infection in mice. This approach offers potential for prophylaxis and/or therapy against drug-resistant strains of K. pneumoniae.