Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-microg p.o. dose of QS-21, whereas the 250-microg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-gamma (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4(-/-) mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.     

Induction of Systemic and Mucosal Immune Responses to Human Immunodeficiency Virus Type 1 by a DNA Vaccine Formulated with QS-21 Saponin Adjuvant via Intramuscular and Intranasal Routes

Induction of mucosal and cell-mediated immunity is critical for development of an effective vaccine against human immunodeficiency virus (HIV). We compared intramuscular and intranasal immunizations with a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant for augmentation of the systemic and mucosal immune responses to HIV-1 in a murine model. Vaccination via the two routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Intestinal secretory IgA production and cytolytic activity of the mesenteric lymph node cells are preferentially elicited by intranasal immunization, and QS-21 augmented these activities as well. This adjuvant augmented production of interleukin-2 (IL-2) and gamma interferon (IFN-γ) associated with decrease in IL-4 synthesis by antigen-restimulated splenocytes. The serum immunoglobulin subtype profile showed a dominant IgG2a response and less strong IgG1 and IgE production in a QS-21 dose-dependent manner. As expected, enhancements of humoral and cell-mediated immune responses by QS-21 were abrogated by treatment with anti-IL-2 and anti-IFN-γ monoclonal antibodies. These results suggest that the intranasal route of DNA immunization is more efficient than the intramuscular route in inducing mucosal immunity mediated by sIgA and mesenteric lymphocytes. Furthermore, QS-21 is able to act as a mucosal adjuvant in DNA vaccination and demonstrates its immunomodulatory property via stimulation of the Th1 subset. This study emphasizes the importance of the route of immunization and the use of an adjuvant for effective DNA vaccination against HIV-1.     

Influence of adjuvants in inducing immune responses to different epitopes included in a multiepitope,multivalent, multistage Plasmodium falciparum candidate vaccine (FALVAC-1) in outbred mice

FALVAC-1, a vaccine against Plasmodium falciparum was developed by joining 21 epitopes from P. falciparum vaccine antigens and an universal T helper epitope from tetanus toxoid. Since adjuvants influence different aspects of immune responses, in this study we investigated the effect of four adjuvants aluminum hydroxide (alum), nonionic copolymer adjuvant P1005 (water-in-oil emulsion), CpG oligodeoxynucleotides (ODN), and QS-21 in eliciting immune responses in outbred mice. QS-21 and copolymer adjuvants were the best formulations in inducing higher and long-lasting antibody titers to the whole vaccine compared to alum and CpG. QS-21 was the only adjuvant to elicit predominantly IgG2a response and antibodies reactive with all epitopes incorporated in the vaccine construct. Vaccine elicited antibodies recognized sporozoites and asexual blood-stage parasites. FALVAC-1 immunized mice induced lymphoproliferative and IFN-gamma response to the vaccine. QS-21 and CpG adjuvants were able to elicit T proliferative responses to 20 of the 22 epitopes in the vaccine. In conclusion, this study demonstrated that with suitable adjuvant such as QS-21, it is possible to elicit immune responses to most of the epitopes included in the FALVAC-1 vaccine.     

Brugia malayi: comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model

Immunization of jirds with Bm-alt-2 elicited partial protection against challenge infection with the filarial parasite Brugia malayi. In this study, we initially compared the protective immune responses elicited following immunization with recombinant Bm-ALT-2 protein regimen and Bm-alt-2 DNA regimen. These studies showed that protein vaccination conferred approximately 75% protection compared to DNA vaccination that conferred only 57% protection. Analysis of the protective immune responses showed that the protein immunization promoted a Th2-biased response with an increase in IL-4, IL-5 and IgG1 responses, whereas, the DNA vaccine promoted a Th1-biased response with profound IFN-gamma and IgG2a responses. Since protein vaccination gave better results than DNA vaccination, we then wanted to evaluate whether a prime-boost vaccination that combined DNA prime and protein boost will significantly increase the protective responses induced by the protein vaccine. Our results suggest that prime-boost vaccination had no added advantage and was comparatively less effective (64% protection) than the Bm-ALT-2 protein alone vaccination. Prime boost vaccination generated mixed Th1/Th2 responses with a slightly diminished Th2 responses compared to protein vaccination. Thus, our results suggest that Bm-ALT-2 protein vaccination regimen may be slightly better than prime-boost vaccine regimen and the mechanism of protection appears to be largely mediated by a Th2-biased response.     

Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine.

The adjuvant activity of a single highly purified saponin from the soap bark tree Quillaja saponaria was evaluated by using it as a component in an experimental vaccine containing rHIV-1 envelope protein (HIV-1 160D) adsorbed to alum. BALB/c mice immunized with experimental vaccine formulations containing the saponin adjuvant QS-21 produced significantly higher titers of antibodies than mice vaccinated with only the alum-adsorbed HIV-1 160D. Potent amnestic antibody responses to HIV-1 viral proteins were also induced. Ag-specific proliferative responses to recombinant proteins and to three variants of HIV-1 were significantly increased using QS-21 as an adjuvant. Alum-adsorbed HIV-1 160D failed to induce measurable proliferative responses to inactivated HIV-1 viruses, but group-specific proliferative responses were raised when the QS-21 adjuvant was used in the vaccine formulation. MHC class I restricted CTL specific for the immunodominant V-3 loop were induced but only when the QS-21 adjuvant was included in the vaccine formulation. The production of serine esterase by Ag-activated splenic mononuclear cells, indicating the maturation of precursor CTL, was used as a secondary measure of CTL activity, and this response was also increased. The specificity of antibody responses was not significantly broadened using QS-21; the adjuvant increased the immune recognition of epitopes throughout the HIV-1 glycoprotein 160. However, the specificity of the proliferation and serine esterase responses was broadened, suggesting that the QS-21 augmented cell-mediated immune responses specific for epitopes outside of the V-3 loop. Additionally, the QS-21 adjuvant appeared to induce recognition of weakly immunogenic epitopes that were not recognized using only alum-adsorbed HIV-1 160D. The ability of QS-21 to augment both antibody and cell-mediated immune responses suggests that this adjuvant could be a valuable component in subunit vaccines.     

Recombinant Sendai virus induces T cell immunity against respiratory syncytial virus that is protective in the absence of antibodies

Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and a vaccine is highly desirable. The fusion (F) protein of RSV is an important vaccine target, but the contribution of F-specific T cells to successful vaccination remains unclear. We studied the immune response to vaccination of mice with a recombinant Sendai virus expressing RSV F (rSeV F). rSeV F induced protective neutralizing antibody and RSV F-specific CTL responses. T cell immunity was stronger than that induced by recombinant vaccinia virus (rVV F), a well characterized reference vector. Vaccination of antibody-deficient mice showed that vaccine-induced RSV F-specific T cells were sufficient for protective immunity. rSeV F induced T cell immunity in the presence of neutralizing antibodies, which did not impair the vaccine response. Although the F protein only contains a subdominant CTL epitope, vaccination with rSeV F is sufficient to induce protective T cell immunity against RSV in mice.     

Intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein induces strong mucosal immune responses and provides long-term protection against SARS-CoV infection

We have previously reported that a subunit protein vaccine based on the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein and a recombinant adeno-associated virus (rAAV)-based RBD (RBD-rAAV) vaccine could induce highly potent neutralizing Ab responses in immunized animals. In this study, systemic, mucosal, and cellular immune responses and long-term protective immunity induced by RBD-rAAV were further characterized in a BALB/c mouse model, with comparison of the i.m. and intranasal (i.n.) routes of administration. Our results demonstrated that: 1) the i.n. vaccination induced a systemic humoral immune response of comparable strength and shorter duration than the i.m. vaccination, but the local humoral immune response was much stronger; 2) the i.n. vaccination elicited stronger systemic and local specific cytotoxic T cell responses than the i.m. vaccination, as evidenced by higher prevalence of IL-2 and/or IFN-gamma-producing CD3+/CD8+ T cells in both lungs and spleen; 3) the i.n. vaccination induced similar protection as the i.m. vaccination against SARS-CoV challenge in mice; 4) higher titers of mucosal IgA and serum-neutralizing Ab were associated with lower viral load and less pulmonary pathological damage, while no Ab-mediated disease enhancement effect was observed; and 5) the vaccination could provide long-term protection against SARS-CoV infection. Taken together, our findings suggest that RBD-rAAV can be further developed into a vaccine candidate for prevention of SARS and that i.n. vaccination may be the preferred route of administration due to its ability to induce SARS-CoV-specific systemic and mucosal immune responses and its better safety profile.     

Manipulation of immune responses to Mycobacterium bovis by vaccination with IL-2- and IL-18-secreting recombinant bacillus Calmette Guerin

Bacillus Calmette Guerin (BCG) has been reported to show variable efficacy as a vaccine against tuberculosis. We demonstrated that the secretion of biologically active IL-2 (rBCG/IL-2),but not IL-18 (rBCG/IL-18), by BCG improves its ability to induce and maintain a strong type 1 immune response in BALB/c mice. rBCG/IL-2 induced significantly higher Ag-specific proliferative responses, high IFN-gamma production and serum titres of IgG2a 16 weeks after vaccination. This immune profile was correlated to an increased rate of clearance of non-pathogenic mycobacteria (live BCG delivered intranasally). Surprisingly, however,this strong type 1 immune profile induced no greater protective immunity against aerosol challenge with virulent Mycobacterium bovis than that induced by normal BCG (nBCG). By comparison,vaccination with rBCG/IL-18 was found to induce significantly less IFN-gamma production in splenic lymphocytes than nBCG.This impaired induction of IFN-gamma was correlated to a significantly lower protective efficacy against M. bovis challenge, as compared to nBCG. The data suggest that manipulation of the immune response to tuberculosis and tuberculosis vaccines will require a more complete understanding of the factors that are important in generating a protective immune response.     

Design,synthesis, and immunologic evaluation of vaccine adjuvant conjugates based on QS-21 and tucaresol

Immunoadjuvants are used to potentiate the activity of modern subunit vaccines that are based on molecular antigens. An emerging approach involves the combination of multiple adjuvants in a single formulation to achieve optimal vaccine efficacy. Herein, to investigate such potential synergies, we synthesized novel adjuvant conjugates based on the saponin natural product QS-21 and the aldehyde tucaresol via chemoselective acylation of an amine at the terminus of the acyl chain domain in QS saponin variants. In a preclinical mouse vaccination model, these QS saponin–tucaresol conjugates induced antibody responses similar to or slightly higher than those generated with related QS saponin variants lacking the tucaresol motif. The conjugates retained potent adjuvant activity, low toxicity, and improved activity–toxicity profiles relative to QS-21 itself and induced IgG subclass profiles similar to those of QS-21, indicative of both Th1 cellular and Th2 humoral immune responses. This study opens the door to installation of other substituents at the terminus of the acyl chain domain to develop additional QS saponin conjugates with desirable immunologic properties.     

Cyclooxygenase 2 inhibition promotes IFN-gamma-dependent enhancement of antitumor responses

In previous studies, we demonstrated an immune suppressive network in non-small cell lung cancer that is due to overexpression of tumor cyclooxygenase 2 (COX-2). In this study, we assessed the vaccination response to tumor challenge following either pharmacological or genetic inhibition of COX-2 in a murine lung cancer model. Treatment of naive mice with the COX-2 inhibitor, SC-58236, skewed splenocytes toward a type 1 cytokine response, inducing IFN-gamma, IL-12, and IFN-gamma-inducible protein 10, whereas the type 2 cytokines IL-4, IL-5, and IL-10 remained unaltered. Fifty percent of mice receiving SC-58236 and an irradiated tumor cell vaccine completely rejected tumors upon challenge. Those mice that did form tumors following challenge demonstrated a reduced tumor growth. In contrast, all mice either vaccinated with irradiated tumor cells alone or receiving SC-58236 alone showed progressive tumor growth. Studies performed in CD4 and CD8 knockout mice revealed a requirement for the CD4 T lymphocyte subset for the complete rejection of tumors. To determine the role of host COX-2 expression on the vaccination responses, studies were performed in COX-2 gene knockout mice. Compared with control littermates, COX-2(-/-) mice showed a significant tumor growth reduction, whereas heterozygous COX-2(-/+) mice had an intermediate tumor growth reduction following vaccination. In vivo depletion of IFN-gamma abrogated the COX-2 inhibitor-mediated enhancement of the vaccination effect. These findings provide a strong rationale for additional evaluation of the capacity of COX-2 inhibitors to enhance vaccination responses against cancer.     

Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles

The recognition that rotaviruses are the major cause of life-threatening diarrheal disease and significant morbidity in young children has focused efforts on disease prevention and control of these viruses. Although the correlates of protection in children remain unclear, some studies indicate that serotype-specific antibody is important. Based on this premise, current live attenuated reassortant rotavirus vaccines include the four predominant serotypes of virus. We are evaluating subunit rotavirus vaccines, 2/6/7-VLPs and 2/4/6/7-VLPs, that contain only a single VP7 of serotype G1 or G3. In mice immunized parenterally twice, G3 virus-like particles (VLPs) induced a homotypic, whereas G1 VLPs induced a homotypic and heterotypic (G3) serum neutralizing immune response. Administration of three doses of G1 or G3 VLPs induced serum antibodies that neutralized five of seven different serotype test viruses. The inclusion of VP4 in the VLPs was not essential for the induction of heterotypic neutralizing antibody in mice. To confirm these results in another species, rabbits were immunized parenterally with two doses of 2/4/6/7-VLPs containing a G3 or G1 VP7, sequentially with G3 VLPs followed by G1 (G3/G1) VLPs, or with live or psoralen-inactivated SA11. High-titer homotypic serum neutralizing antibody was induced in all rabbits, and low-level heterotypic neutralizing antibody was induced in a subset of rabbits. The rabbits immunized with the G1 or G3/G1 VLPs in QS-21 were challenged orally with live G3 ALA rotavirus. Protection levels were similar in rabbits immunized with homotypic G3 2/4/6/7-VLPs, heterotypic G1 2/4/6/7-VLPs, or G3/G1 2/4/6/7-VLPs. Therefore, G1 2/4/6/7-VLPs can induce protective immunity against a live heterotypic rotavirus challenge in an adjuvant with potential use in humans. Following challenge, broad serum heterotypic neutralizing antibody responses were detected in rabbits parenterally immunized with G1, G3/G1, or G3 VLPs but not with SA11. Immunization with VLPs may provide sufficient priming of the immune system to induce protective anamnestic heterotypic neutralizing antibody responses upon subsequent rotavirus infection. Therefore, a limited number of serotypes of VLPs may be sufficient to provide a broadly protective subunit vaccine.     

IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.     

Immunization of Rhesus monkeys with a SialylTn–mAb17-1A conjugate vaccine co-formulated with QS-21 induces a temporary systemic cytokine release and NK cytotoxicity against tumor cells

Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn carbohydrate antigen, are frequently over-expressed on cancer cells and provide potential targets for cancer vaccination. Immunization of Rhesus monkeys with SialylTn coupled to a highly immunogenic carrier molecule and formulated on aluminum hydroxide induced a strong immune response against the carrier protein but only a moderate IgM immune response against the SialylTn carbohydrate antigen. Co-formulation with QS-21 adjuvant dramatically enhanced the anti-SialylTn immune response and resulted in a SialylTn-specific IgG switch. The kinetics of the carbohydrate-specific IgG response correlated with a temporary release of cytokines such as IFNγ, IL-2, IL-1β, TNFα and GM-CSF which was measurable in the immune serum by xMAP Multiplex technology. Furthermore, tumor cell killing by activated natural killer cells was induced. These data demonstrate that immunization with a tumor-associated carbohydrate antigen in a highly immunogenic formulation results in a temporary release of type 1 cytokines which may be required for the induction of a specific IgG immune response against the carbohydrate antigen as well as for activation of effector cells against tumor cells.     

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.     

Subunit Rotavirus Vaccine Administered Parenterally to Rabbits Induces Active Protective Immunity

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund’s adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 10³ 50% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P < 0.001, r = 0.55; Pearson’s correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund’s adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund’s adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund’s adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine.     

The immunomodulating properties of human respiratory syncytial virus and immunostimulating complexes containing Quillaja saponin components QH-A, QH-C and ISCOPREP703

A successful vaccine against human RSV (HRSV) is likely to induce a Th1 or a balanced Th1/TH2 cytokine response. We tested a panel of HRSV immunostimulating complexes (ISCOMs) containing different Quillaja saponin fractions (QH-A, QH-C, and 703: a mixture of 70% QH-A and 30% QH-C) with different immunological properties for their capacity of inducing innate and acquired immune responses. The HRSV 703 ISCOMs induced the strongest innate and acquired immune responses, followed by RSV QH-C and QH-A ISCOMs. All three formulations induced various degrees of Th1 bias response with prominent production of IFN-gamma being 10-50 times higher than that of IL-4 and IL-5. The HRSV specific IgG isotype profile correlated with the predominant secretion of Th1 cytokines, with strong induction of IgG2a antibodies. The 703 ISCOMs induced the most pronounced Th1 profile followed by QH-C and QH-A ISCOMs. The high incorporation of F protein in these ISCOMs compared to G protein combined with the Th1 biased nature of ISCOM are likely to be the causes to promote a Th1 type of profile. The prospect to formulate an RSV ISCOM formulation with an optimal Th1/Th2 balance is in reach particularly in view of the versatile properties of the ISCOM concept.     

Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization

A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors.Recombinant adenovirus (rAd)-based vectors are currently under investigation in a variety of gene therapy and T-cell-based vaccine clinical trials. There are more than 370 such ongoing clinical trials for broad applications, including infectious diseases and cancer therapy (http://www.wiley.co.uk/genetherapy/clinical/). Based on supportive data from nonhuman primate studies, rAd-based vectors have been developed and tested in human clinical trials to deliver human immunodeficiency virus (HIV-1) gene products that stimulate HIV-specific immune responses. Preexisting immunity to Ad serotype 5 (Ad5), from which most vectors are derived, is common in humans. Though neutralizing antibodies to Ad5 may reduce the immunogenicity of Ad5-based vectors in animal models (16), their effect on immunity in subjects with previous Ad5 infection is poorly understood. In the STEP trial, which tested a Merck rAd5 vaccine encoding HIV-1 Gag, Pol, and Nef, vaccination failed to show protection, either by lowering viral load or by decreasing acquisition of infection (3, 9, 12, 21). Furthermore, the possibility was raised that subjects with preexisting neutralizing antibodies from natural Ad5 infection may have carried an increased risk of HIV infection after vaccination. Thus, understanding the nature and immune effects of Ad5 seropositivity in humans is important to the development of vaccines against AIDS and other diseases.Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis in humans, and exposure before adolescence is common in human populations (19). Such exposure stimulates both innate and adaptive immune responses that generate neutralizing antibodies and virus-specific T-cell responses (6). These antibodies can also synergize with each other to achieve maximum viral neutralization (7, 22). The capsid protein specificity of Ad5 neutralizing antibodies has been reported for humans following administration of rAd5 gene therapy vectors for advanced liver or lung cancer (7, 10). However, results were presented solely for antibodies induced by administration of rAd5. One report has assessed Ad5 neutralizing antibodies with a healthy human population that was Ad5 seropositive from natural exposure to the virus (18). The median titer of the population was presented, but the frequency of protein-specific neutralizing antibody has not been defined for humans.Here we describe the first report of the natural frequency and effect on immunization of neutralizing antibodies specific for different Ad capsid proteins in human subjects. We address the fundamental mechanisms of how humans generate neutralizing antibodies to a common cold virus that is in widespread use as a vector for gene therapy and vaccines. Such mechanisms may also be applicable to other nonenveloped viruses, including adeno-associated viruses and other viruses containing multiple envelope surface proteins, like influenza. To analyze the contribution of anti-capsid antibodies to neutralization by different human serum samples, wild-type and chimeric vectors were utilized. For example, a rAd type 5 (rAd5) vector with a fiber derived from Ad35 fiber (rAd5 F35) can be used to analyze the anti-Ad5 capsid response independent of fiber. Conversely, a rAd35 vector with a fiber transposed from Ad5 can determine the specificity of neutralization mediated by the Ad5 fiber. Using these vectors, we have analyzed human serum samples from two HIV vaccine clinical trials, VRC 006 and HVTN 204, in which a single-dose rAd5 vaccine alone and a three-dose DNA prime/single dose rAd5 boost vaccine encoding HIV-1 Env A,B, and C; Gag; and Pol, respectively, were administered. Thus, we sought to characterize the specificity of rAd5 neutralizing antibodies in Ad5-immune subjects and to determine their effect on immune responses elicited by vaccination.     

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.     

T cell responses to whole SARS coronavirus in humans

Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-gamma ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8(+) T cell responses were more frequent and of a greater magnitude than CD4(+) T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27(+)/CD45RO(+)) with a significantly higher frequency of polyfunctional CD4(+) T cells producing IFN-gamma, TNF-alpha, and IL-2, and CD8(+) T cells producing IFN-gamma, TNF-alpha, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation.     

Allogeneic retrovirally transduced, IL-2- and IFN-gamma-secreting cancer cell vaccine in patients with hormone refractory prostate cancer--a phase I clinical trial

BACKGROUND: The purpose of this vaccine study was to determine the safety and feasibility of vaccination with an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and to evaluate the efficacy of inducing tumor-specific immune responses in HLA-A2-matched patients with hormone refractory prostate cancer (HRPC). METHODS: In a dose-escalating phase I study, HLA-A2-matched HRPC patients received four vaccinations of irradiated allogeneic LNCaP cells retrovirally transduced to secrete IL-2 and IFN-gamma at study day 1, 15, 29 and 92 and subsequently every 91 days unless tumor progression was evident. RESULTS: Three patients receiving the first dose level (7.5 million cells) showed no evidence of dose-limiting toxicity or vaccine-related adverse events including autoimmunity. One of three patients receiving the second dose level (15 million cells) developed a transient self-limiting grade 3 local injection site reaction (ulceration) after the eighth vaccination. Vaccine-induced immune responses against a broad array of prostate tumor associated antigens were detected in all six patients. Two of the three patients receiving the higher dose showed a decline in serum prostate-specific antigen (PSA) values of more than 50%, with one patient remaining on protocol for 3 years. CONCLUSIONS: Immunisation with the allogeneic LNCaP/IL-2/IFN-gamma vaccine is safe and feasible without any dose-limiting toxicity or autoimmunity. A 50% PSA decline was achieved in two of the six patients. This encouraging data provides the scientific rationale for further investigation of the vaccine in a phase II trial.