Astragalus Root and Elderberry Fruit Extracts Enhance the IFN-β Stimulatory Effects of Lactobacillus acidophilus in Murine-Derived Dendritic Cells

Many foods and food components boost the immune system, but little data are available regarding the mechanisms by which they do. Bacterial strains have disparate effects in stimulating the immune system. Indendritic cells, the gram-negative bacteria Escherichia coli upregulates proinflammatory cytokines, whereas gram-positive Lactobacillus acidophilus induces a robust interferon (IFN)-β response. The immune-modulating effects of astragalus root and elderberry fruit extracts were examined in bone marrow-derived murine dendritic cells that were stimulated with L. acidophilus or E. coli. IFN-β and other cytokines were measured by ELISA and RT-PCR. Endocytosis of fluorescence-labeled dextran and L. acidophilus in the presence of elderberry fruit or astragalus root extract was evaluated in dendritic cells. Our results show that both extracts enhanced L. acidophilus-induced IFN-β production and slightly decreased the proinflammatory response to E. coli. The enhanced IFN-β production was associated with upregulation of toll-like receptor 3 and to a varying degree, the cytokines IL-12, IL-6, IL-1β and TNF-α. Both extracts increased endocytosis in immature dendritic cells, and only slightly influenced the viability of the cells. In conclusion, astragalus root and elderberry fruit extracts increase the IFN-β inducing activity of L. acidophilus in dendritic cells, suggesting that they may exert antiviral and immune-enhancing activity.     

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4⁺ T-cell epitopes; one T-cell epitope located at E_349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E_313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.     

Vaccination with Leishmania infantum Acidic Ribosomal P0 but Not with Nucleosomal Histones Proteins Controls Leishmania infantum Infection in Hamsters

Background

Several intracellular Leishmania antigens have been identified in order to find a potential vaccine capable of conferring long lasting protection against Leishmania infection. Histones and Acid Ribosomal proteins are already known to induce an effective immune response and have successfully been tested in the cutaneous leishmaniasis mouse model. Here, we investigate the protective ability of L. infantum nucleosomal histones (HIS) and ribosomal acidic protein P0 (LiP0) against L. infantum infection in the hamster model of visceral leishmaniasis using two different strategies: homologous (plasmid DNA only) or heterologous immunization (plasmid DNA plus recombinant protein and adjuvant).

Methodology/Principal Findings

Immunization with both antigens using the heterologous strategy presented a high antibody production level while the homologous strategy immunized group showed predominantly a cellular immune response with parasite load reduction. The pcDNA-LiP0 immunized group showed increased expression ratio of IFN-γ/IL-10 and IFN-γ/TGF-β in the lymph nodes before challenge. Two months after infection hamsters immunized with the empty plasmid presented a pro-inflammatory immune response in the early stages of infection with increased expression ratio of IFN-γ/IL-10 and IFN-γ/TGF-β, whereas hamsters immunized with pcDNA-HIS presented an increase only in the ratio IFN-γ/ TGF-β. On the other hand, hamsters immunized with LiP0 did not present any increase in the IFN-γ/TGF-β and IFN-γ/IL-10 ratio independently of the immunization strategy used. Conversely, five months after infection, hamsters immunized with HIS maintained a pro-inflammatory immune response (ratio IFN-γ/ IL-10) while pcDNA-LiP0 immunized hamsters continued showing a balanced cytokine profile of pro and anti-inflammatory cytokines. Moreover we observed a significant reduction in parasite load in the spleen, liver and lymph node in this group compared with controls.

Conclusions/Significance

Our results suggest that vaccination with L. infantum LiP0 antigen administered in a DNA formulation could be considered a potential component in a vaccine formulation against visceral leishmaniasis.     

Role of 4-1BB Receptor in the Control Played by CD8+ T Cells on IFN-γ Production by Mycobacterium tuberculosis Antigen-Specific CD4+ T Cells

Background

Antigen-specific IFN-γ producing CD4⁺ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-γ production without affecting protective IFN-γ is a challenge in tuberculosis research.

Methods and Findings

Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4⁺ T cell-mediated IFN-γ response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-γ response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8⁺ T cells which suppressed IFN-γ-secreting CD4⁺ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-γ responses by CD4⁺ T cells in protein-boosted mice without affecting the low protective IFN-γ-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8⁺ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-γ inhibition did not require soluble IL-10, TGF-β, XCL-1 and MIP-1β. In vivo Ag85B stimulation induced 4-1BB expression on CD8⁺ T cells and in vivo 4-1BB ligation reduced the activation, IFN-γ production and expansion of Ag85B-specific CD4⁺ T cells of DNA-primed and protein-boosted mice.

Conclusion/Significance

Antigen-specific suppressor CD8⁺ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-γ-secreting CD4⁺ T cells. The selective expression of 4-1BB only on CD8⁺ T cells in mice developing a massive, non-protective IFN-γ response opens novel strategies for intervention in tuberculosis pathology and vaccination through T-cell co-stimulatory-based molecular targeting.     

Vaxfectin Adjuvant Improves Antibody Responses of Juvenile Rhesus Macaques to a DNA Vaccine Encoding the Measles Virus Hemagglutinin and Fusion Proteins

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.m. or 100 μg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 μg of DNA plus Vaxfectin than after 100 μg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.     

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.     

Th1-Th17 Cells Mediate Protective Adaptive Immunity against Staphylococcus aureus and Candida albicans Infection in Mice

We sought to define protective mechanisms of immunity to Staphylococcus aureus and Candida albicans bloodstream infections in mice immunized with the recombinant N-terminus of Als3p (rAls3p-N) vaccine plus aluminum hydroxide (Al(OH₃) adjuvant, or adjuvant controls. Deficiency of IFN-γ but not IL-17A enhanced susceptibility of control mice to both infections. However, vaccine-induced protective immunity against both infections required CD4+ T-cell-derived IFN-γ and IL-17A, and functional phagocytic effectors. Vaccination primed Th1, Th17, and Th1/17 lymphocytes, which produced pro-inflammatory cytokines that enhanced phagocytic killing of both organisms. Vaccinated, infected mice had increased IFN-γ, IL-17, and KC, increased neutrophil influx, and decreased organism burden in tissues. In summary, rAls3p-N vaccination induced a Th1/Th17 response, resulting in recruitment and activation of phagocytes at sites of infection, and more effective clearance of S. aureus and C. albicans from tissues. Thus, vaccine-mediated adaptive immunity can protect against both infections by targeting microbes for destruction by innate effectors.     

Heterologous Prime-Boost Regimens Using rAd35 and rMVA Vectors Elicit Stronger Cellular Immune Responses to HIV Proteins Than Homologous Regimens

We characterized prime-boost vaccine regimens using heterologous and homologous vector and gene inserts. Heterologous regimens offer a promising approach that focuses the cell-mediated immune response on the insert and away from vector-dominated responses. Ad35-GRIN/ENV (Ad35-GE) vaccine is comprised of two vectors containing sequences from HIV-1 subtype A gag, rt, int, nef (Ad35-GRIN) and env (Ad35-ENV). MVA-CMDR (MVA-C), MVA-KEA (MVA-K) and MVA-TZC (MVA-T) vaccines contain gag, env and pol genes from HIV-1 subtypes CRF01_AE, A and C, respectively. Balb/c mice were immunized with different heterologous and homologous vector and insert prime-boost combinations. HIV and vector-specific immune responses were quantified post-boost vaccination. Gag-specific IFN-γ ELISPOT, intracellular cytokine staining (ICS) (CD107a, IFN-γ, TNF-α and IL-2), pentamer staining and T-cell phenotyping were used to differentiate responses to inserts and vectors. Ad35-GE prime followed by boost with any of the recombinant MVA constructs (rMVA) induced CD8+ Gag-specific responses superior to Ad35-GE-Ad35-GE or rMVA-rMVA prime-boost combinations. Notably, there was a shift toward insert-focus responses using heterologous vector prime-boost regimens. Gag-specific central and effector memory T cells were generated more rapidly and in greater numbers in the heterologous compared to the homologous prime-boost regimens. These results suggest that heterologous prime-boost vaccination regimens enhance immunity by increasing the magnitude, onset and multifunctionality of the insert-specific cell-mediated immune response compared to homologous vaccination regimens. This study supports the rationale for testing heterologous prime-boost regimens in humans.     

Interferon-γ Induced by In Vitro Re-Stimulation of CD4+ T-Cells Correlates with In Vivo FMD Vaccine Induced Protection of Cattle against Disease and Persistent Infection

The immune defense against FMDV has been correlated to the antibody mediated component. However, there are occasions when some animals with high virus neutralising (VN) antibody are not protected following challenge and some with low neutralising antibody which do not succumb to disease. The importance of cell mediated immunity in clinical protection is less clear and so we investigated the source and production of interferon-gamma (IFN-γ) in re-stimulated whole blood of FMDV immunized cattle and its correlation to vaccine induced protection and FMDV persistence. We were able to show a positive correlation between IFN-γ response and vaccine induced protection as well as reduction of long term persistence of FMD virus. When combining this IFN-γ response in re-stimulated blood with virus neutralizing antibody titer in serum on the day of challenge, a better correlation of vaccine-induced protection with IFN-γ and VN antibody was predicted. Our investigations also showed that CD4+ T-cells are the major proliferating phenotype and IFN-γ producing cells.     

In Vitro and In Vivo Studies for Assessing the Immune Response and Protection-Inducing Ability Conferred by Fasciola hepatica-Derived Synthetic Peptides Containing B- and T-Cell Epitopes

Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05) and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L⁺ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance.     

Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen

The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.     

Quantitative PCR Evaluation of Cellular Immune Responses in Kenyan Children Vaccinated with a Candidate Malaria Vaccine

Background

The T-cell mediated immune response plays a central role in the control of malaria after natural infection or vaccination. There is increasing evidence that T-cell responses are heterogeneous and that both the quality of the immune response and the balance between pro-inflammatory and regulatory T-cells determines the outcome of an infection. As Malaria parasites have been shown to induce immunosuppressive responses to the parasite and non-related antigens this study examined T-cell mediated pro-inflammatory and regulatory immune responses induced by malaria vaccination in children in an endemic area to determine if these responses were associated with vaccine immunogenicity.

Methods

Using real–time RT- PCR we profiled the expression of a panel of key markers of immunogenecity at different time points after vaccination with two viral vector vaccines expressing the malaria TRAP antigen (FP9-TRAP and MVA-TRAP) or following rabies vaccination as a control.

Principal Findings

The vaccine induced modest levels of IFN-γ mRNA one week after vaccination. There was also an increase in FoxP3 mRNA expression in both TRAP stimulated and media stimulated cells in the FFM ME-TRAP vaccine group; however, this may have been driven by natural exposure to parasite rather than by vaccination.

Conclusion

Quantitative PCR is a useful method for evaluating vaccine induced cell mediated immune responses in frozen PBMC from children in a malaria endemic country. Future studies should seek to use vaccine vectors that increase the magnitude and quality of the IFN-γ immune response in naturally exposed populations and should monitor the induction of a regulatory T cell response.     

Early IFN-Gamma Production after YF 17D Vaccine Virus Immunization in Mice and Its Association with Adaptive Immune Responses

Yellow Fever vaccine is one of the most efficacious human vaccines ever made. The vaccine (YF 17D) virus induces polyvalent immune responses, with a mixed T_H1/T_H2 CD4⁺ cell profile, which results in robust T CD8⁺ responses and high titers of neutralizing antibody. In recent years, it has been suggested that early events after yellow fever vaccination are crucial to the development of adequate acquired immunity. We have previously shown that primary immunization of humans and monkeys with YF 17D virus vaccine resulted in the early synthesis of IFN-γ. Herein we have demonstrated, for the first time that early IFN-γ production after yellow fever vaccination is a feature also of murine infection and is much more pronounced in the C57BL/6 strain compared to the BALB/c strain. Likewise, in C57BL/6 strain, we have observed the highest CD8⁺ T cells responses as well as higher titers of neutralizing antibodies and total anti-YF IgG. Regardless of this intense IFN-γ response in mice, it was not possible to see higher titers of IgG2a in relation to IgG1 in both mice lineages. However, IgG2a titers were positively correlated to neutralizing antibodies levels, pointing to an important role of IFN-γ in eliciting high quality responses against YF 17D, therefore influencing the immunogenicity of this vaccine.     

IFN-α as an Adjuvant for Adenovirus-Vectored FMDV Subunit Vaccine through Improving the Generation of T Follicular Helper Cells

IFN-α exhibits either direct antiviral effects or distinct immunomodulatory properties, which was identified as a ‘natural immune adjuvant’ for both the innate and the adaptive immune responses. Here we have investigated the effects of IFN-α as an adjuvant on the generation of T follicular helper (Tfh) cells and antigen-specific antibody responses. The data showed that adenoviral vectors co-expressing FMDV VP1 proteins and porcine IFN-α potently enhanced the generation of Tfh cells, the secretion of IL-21 protein and the expression of Bcl-6 mRNA, compared with adenoviral vectors sole expressing VP1 alone. Additionally, IFN-α substantial increased the number of germinal-center (GC) B cells and formation of GCs. Furthermore, IFN-α enhanced the antibody response, as shown by increased production of all IgG and subclasses of IgG1 and IgG2a. Thus, our results revealed the potent adjuvant activity of IFN-α which enhanced the generation of Tfh cells and regulated the humoral immunity by promoting germinal-center reactions and antibody responses.     

Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial Reveals an Association of Nonspecific Interferon-γ Secretion with Increased HIV-1 Infection Risk: A Cohort-Based Modeling Study

Background

Elevated risk of HIV-1 infection among recipients of an adenovirus serotype 5 (Ad5)-vectored HIV-1 vaccine was previously reported in the Step HIV-1 vaccine efficacy trial. We assessed pre-infection cellular immune responses measured at 4 weeks after the second vaccination to determine their roles in HIV-1 infection susceptibility among Step study male participants.

Methods

We examined ex vivo interferon-γ (IFN-γ) secretion from peripheral blood mononuclear cells (PBMC) using an ELISpot assay in 112 HIV-infected and 962 uninfected participants. In addition, we performed flow cytometric assays to examine T-cell activation, and ex vivo IFN-γ and interleukin-2 secretion from CD4⁺ and CD8⁺ T cells. We accounted for the sub-sampling design in Cox proportional hazards models to estimate hazard ratios (HRs) of HIV-1 infection per 1-log_e increase of the immune responses.

Findings

We found that HIV-specific immune responses were not associated with risk of HIV-1 infection. However, each 1-log_e increase of mock responses measured by the ELISpot assay (i.e., IFN-γ secretion in the absence of antigen-specific stimulation) was associated with a 62% increase of HIV-1 infection risk among vaccine recipients (HR = 1.62, 95% CI: (1.28, 2.04), p<0.001). This association remains after accounting for CD4⁺ or CD8⁺ T-cell activation. We observed a moderate correlation between ELISpot mock responses and CD4⁺ T-cells secreting IFN-γ (ρ = 0.33, p = 0.007). In addition, the effect of the Step vaccine on infection risk appeared to vary with ELISpot mock response levels, especially among participants who had pre-existing anti-Ad5 antibodies (interaction p = 0.04).

Conclusions

The proportion of cells, likely CD4⁺ T-cells, producing IFN-γ without stimulation by exogenous antigen appears to carry information beyond T-cell activation and baseline characteristics that predict risk of HIV-1 infection. These results motivate additional investigation to understand the potential link between IFN-γ secretion and underlying causes of elevated HIV-1 infection risk among vaccine recipients in the Step study.     

Approaches towards DNA Vaccination against a Skin Ciliate Parasite in Fish

Rainbow trout (Oncorhynchus mykiss) were immunized with plasmid DNA vaccine constructs encoding selected antigens from the parasite Ichthyophthirius multifiliis. Two immobilization antigens (I-ags) and one cysteine protease were tested as genetic vaccine antigen candidates. Antigenicity was evaluated by immunostaining of transfected fish cells using I-ag specific mono- and polyclonal antibodies. I. multifiliis specific antibody production, regulation of immune-relevant genes and/or protection in terms of parasite burden or mortality was measured to evaluate the induced immune response in vaccinated fish. Apart from intramuscular injection, needle free injection and gene gun delivery were tested as alternative administration techniques. For the I-ags the complement protein fragment C3d and the termini of the viral haemorrhagic septicaemia virus glyco(G)protein (VHSV G) were tested as opsonisation and cellular localisation mediators, respectively, while the full length viral G protein was tested as molecular adjuvant. Expression of I-ags in transfected fish cells was demonstrated for several constructs and by immunohistochemistry it was possible to detect expression of a secreted form of the Iag52B in the muscle cells of injected fish. Up-regulations of mRNA coding for IgM, MHC I, MHC II and TCR β, respectively, were observed in muscle tissue at the injection site in selected trials. In the spleen up-regulations were found for IFN-γ and IL-10. The highest up-regulations were seen following co-administration of I-ag and cysteine protease plasmid constructs. This correlated with a slight elevation of an I. multifiliis specific antibody response. However, in spite of detectable antigen expression and immune reactions, none of the tested vaccination strategies provided significant protection. This might suggest an insufficiency of DNA vaccination alone to trigger protective mechanisms against I. multifiliis or that other or additional parasite antigens are required for such a vaccine to be successful.     

Attrition of T-Cell Functions and Simultaneous Upregulation of Inhibitory Markers Correspond with the Waning of BCG-Induced Protection against Tuberculosis in Mice

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live attenuated vaccine. However, the correlates of protection and waning of its immunity against tuberculosis is poorly understood. In this study, we correlated the longitudinal changes in the magnitude and functional quality of CD4⁺ and CD8⁺ T-cell response over a period of two years after mucosal or parenteral BCG vaccination with the strength of protection against Mycobacterium tuberculosis in mice. The BCG vaccination-induced CD4⁺ and CD8⁺ T cells exhibited comparable response kinetics but distinct functional attributes in-terms of IFN-γ, IL-2 and TNF-α co-production and CD62L memory marker expression. Despite a near life-long BCG persistence and the induction of enduring CD4⁺ T-cell responses characterized by IFN-γ and/or TNF-α production with comparable protection, the protective efficacy waned regardless of the route of vaccination. The progressive decline in the multifactorial functional abilities of CD4⁺ and CD8⁺ T cells in-terms of type-1 cytokine production, proliferation and cytolytic potential corresponded with the waning of protection against M. tuberculosis infection. In addition, simultaneous increase in the dysfunctional and terminally-differentiated T cells expressing CTLA-4, KLRG-1 and IL-10 during the contraction phase of BCG-induced response coincided with the loss of protection. Our results question the empirical development of BCG-booster vaccines and emphasize the pursuit of strategies that maintain superior T-cell functional capacity. Furthermore, our results underscore the importance of understanding the comprehensive functional dynamics of antigen-specific T-cell responses in addition to cytokine polyfunctionality in BCG-vaccinated hosts while optimizing novel vaccination strategies against tuberculosis.     

Lower Levels of Gamma Interferon Expressed by a Pseudotyped Single-Cycle Simian Immunodeficiency Virus Enhance Immunogenicity in Rats

A vaccine for human immunodeficiency virus (HIV) infection is desperately needed to control the AIDS pandemic. To address this problem, we constructed single-cycle simian immunodeficiency viruses (SIVs) pseudotyped with the glycoprotein of vesicular stomatitis virus and expressing different levels of gamma interferon (IFN-γ) as a potential vaccine strategy. We previously showed that IFN-γ expression by pseudotyped SIVs does not alter viral single-cycle infectivity. T cells primed with dendritic cells transduced by pseudotyped SIVs expressing high levels of IFN-γ had stronger T-cell responses than those primed with dendritic cells transduced by constructs lacking IFN-γ. In the present study, we tested the immunogenicities of these pseudotyped SIVs in a rat model. The construct expressing low levels of rat IFN-γ (dSIV_LRγ) induced higher levels of cell-mediated and humoral immune responses than the construct lacking IFN-γ (dSIV_R). Rats vaccinated with dSIV_LRγ also had lower viral loads than those vaccinated with dSIV_R when inoculated with a recombinant vaccinia virus expressing SIV Gag-Pol as a surrogate challenge. The construct expressing high levels of IFN-γ (dSIV_HRγ) did not further enhance immunity and was less protective than dSIV_LRγ. In conclusion, the data indicated that IFN-γ functioned as an adjuvant to augment antigen-specific immune responses in a dose- and cell type-related manner in vivo. Thus, fine-tuning of the cytokine expression appears to be essential in designing vaccine vectors expressing adjuvant genes such as the gene for IFN-γ. Furthermore, we provide evidence of the utility of the rat model to evaluate the immunogenicities of single-cycle HIV/SIV recombinant vaccines before initiating studies with nonhuman primate models.     

DNA Vaccination Affords Significant Protection against Feline Immunodeficiency Virus Infection without Inducing Detectable Antiviral Antibodies

To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion in pol (FIVΔRT). In a first experiment, FIVΔRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-γ) DNA. The DNA was administered in four 100-μg doses at 0, 10, and 23 weeks. Immunization with FIVΔRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVΔRT vaccinates than in the controls. Immunization with FIVΔRT in conjunction with IFN-γ gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVΔRT plus IFN-γ and IFN-γ alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines.