Pre-Clinical Development of BCG.HIVA(CAT), an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG

In the past, we proposed to develop a heterologous recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost dual pediatric vaccine platform against transmission of breast milk HIV-1 and Mycobacterium tuberculosis (Mtb). In this study, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVA^CAT expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph (safer) strain of BCG to generate vaccine BCG.HIVA^CAT. All procedures complied with Good Laboratory Practices (GLPs). We demonstrated that the episomal plasmid pJH222.HIVA^CAT was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVA^CAT vaccine strain. The BCG.HIVA^CAT vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific interferon γ-producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVA^CAT and boosted with MVA.HIVA.85A, HIV-1-specific CD8⁺ T-cells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. To assess the biosafety profile of BCG.HIVA^CAT-MVA.HIVA regimen, body mass loss of newborn mice was monitored regularly throughout the vaccination experiment and no difference was observed between the vaccinated and naïve groups of animals. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Second generation immunogens derived from HIV-1 as well as other major pediatric pathogens can be constructed in a similar fashion to prime protective responses soon after birth.     

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8⁺ T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8⁺ T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8⁺ T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8⁺ T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.     

Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.     

Enhancement of the expression of HCV core gene does not enhance core-specific immune response in DNA immunization: advantages of the heterologous DNA prime, protein boost immunization regimen

Background

Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed.

Methods

Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1–98 and 1–173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-μg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 μg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 μg of core aa 1–98 in prime and boost, or with 100 μg of pCMVcoreKozak in prime and 20 μg of core aa 1–98 in boost (III). Antibody response, [³H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization.

Results

Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 ± 0.18, 0.83 ± 0.5, and 13 ± 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-γ and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 10³(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-γ secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regimen that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer ≥ 3 × 10³.

Conclusion

Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regimen that circumvents the negative effects of intracellular core expression.     

Antibodies to gp120 and PD-1 Expression on Virus-Specific CD8+ T Cells in Protection from Simian AIDS

We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4⁺ and CD8⁺ T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8⁺CM9⁺ effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8⁺ T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8⁺ T-cell responses.     

TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA) enhances CD8+ T Cell responses providing protection against Leishmania (Viannia)

Background

Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.

Methodology

Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime – modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease.

Results

Heterologous prime – boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.

Conclusions

Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.     

Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection

Background

Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice.

Methodology/Principal Findings

Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo.

Conclusion

Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.     

One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies,Effector CD4+ T Cells,and IL-17A

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4⁺ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4⁺ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.     

Poly(I:C)/Alum Mixed Adjuvant Priming Enhances HBV Subunit Vaccine-Induced Immunity in Mice When Combined with Recombinant Adenoviral-Based HBV Vaccine Boosting

Background

Virus-specific cellular immune responses play a critical role in virus clearance during acute or chronic HBV infection. Currently, the commercially available HBV vaccine is combined with alum adjuvant, which stimulates mainly Th2 immune responses. Therefore, development of new therapeutic HBV vaccine adjuvants and immune strategies that also promote Th1 and CTL responses is urgently needed.

Methodology/Principal findings

To improve the immunity induced by the novel HBSS1 HBV vaccine, we evaluated the ability of adjuvants, including alum, CpG and polyriboinosinic polyribocytidylic acid [poly(I:C)], to enhance the response when boosted with the recombinant adenoviral vector vaccine rAdSS1. The immune responses to different adjuvant combinations were assessed in C57BL/6 mice by enzyme-linked immunosorbent assay (ELISA), ELISpot and cytokine release assays. Among the combinations tested, a HBV protein particle vaccine with CpG/alum and poly(I:C)/alum priming combinations accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres with a Th1 bias. After boosting with recombinant adenoviral vector vaccine rAdSS1, both groups produced a strong multi-antigen (S and PreS1)-specific cellular immune response. HBSS1 immunisation with poly(I:C)/alum priming also generated high-level CD4⁺ and CD8⁺ T cell responses in terms of Th1 cytokines (IFN-γand IL-2).

Conclusions

The protein-vaccine HBSS1 with mixed poly(I:C)/alum adjuvant priming, followed by a rAdSS1 vaccine boost, maximises specific antibody and Th1-biased cellular immune responses. This regime might prove useful in the development of HBV therapeutic vaccines. Furthermore, this promising strategy might be applied to vaccines against other persistent infections, such as human immunodeficiency virus and tuberculosis.     

A Novel Prime and Boost Regimen of HIV Virus-Like Particles with TLR4 Adjuvant MPLA Induces Th1 Oriented Immune Responses against HIV

HIV virus-like particles (VLPs) present the HIV envelope protein in its native conformation, providing an ideal vaccine antigen. To enhance the immunogenicity of the VLP vaccine, we sought to improve upon two components; the route of administration and the additional adjuvant. Using HIV VLPs, we evaluated sub-cheek as a novel route of vaccine administration when combined with other conventional routes of immunization. Of five combinations of distinct prime and boost sequences, which included sub-cheek, intranasal, and intradermal routes of administration, intranasal prime and sub-cheek boost (IN+SC) resulted in the highest HIV-specific IgG titers among the groups tested. Using the IN+SC regimen we tested the adjuvant VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) + monophosphoryl lipid A (MPLA) at MPLA concentrations of 0, 7.5, 12.5, and 25 μg/dose in combination with our VLPs. Mice that received 12.5 or 25 μg/dose MPLA had the highest concentrations of Env-specific IgG2c (20.7 and 18.4 μg/ml respectively), which represents a Th1 type of immune response in C57BL/6 mice. This was in sharp contrast to mice which received 0 or 7.5 μg MPLA adjuvant (6.05 and 5.68 μg/ml of IgG2c respectively). In contrast to IgG2c, MPLA had minor effects on Env-specific IgG1; therefore, 12.5 and 25 μg/dose of MPLA induced the optimal IgG1/IgG2c ratio of 1.3. Additionally, the percentage of germinal center B cells increased significantly from 15.4% in the control group to 31.9% in the CALV + 25 μg MPLA group. These mice also had significantly more IL-2 and less IL-4 Env-specific CD8⁺ T cells than controls, correlating with an increased percentage of Env-specific central memory CD4⁺ and CD8⁺ T cells. Our study shows the strong potential of IN+SC as an efficacious route of administration and the effectiveness of VLPs combined with MPLA adjuvant to induce Env specific Th1-oriented HIV-specific immune responses.     

Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity

Systemic IL-2 is currently employed in the therapy of several tumor types, but at the price of often severe toxicities. Local vector mediated delivery of IL-2 at the tumor site may enhance local effector cell activity while reducing toxicity. To examine this, a model using CEA-transgenic mice bearing established CEA expressing tumors was employed. The vaccine regimen was a s.c. prime vaccination with recombinant vaccinia (rV) expressing transgenes for CEA and a triad of costimulatory molecules (TRICOM) followed by i.t. boosting with rF-CEA/TRICOM. The addition of intratumoral (i.t.) delivery of IL-2 via a recombinant fowlpox (rF) IL-2 vector greatly enhanced anti-tumor activity of a recombinant vaccine, resulting in complete tumor regression in 70–80% of mice. The anti-tumor activity was shown to be dependent on CD8⁺ cells and NK1.1⁺. Cellular immune assays revealed that the addition of rF-IL-2 to the vaccination therapy enhanced CEA-specific tetramer⁺ cell numbers, cytokine release and CTL lysis of CEA⁺ targets. Moreover, tumor-bearing mice vaccinated with the CEA/TRICOM displayed an antigen cascade, i.e., CD8⁺ T cell responses to two other antigens expressed on the tumor and not the vaccine: wild-type p53 and endogenous retroviral antigen gp70. Mice receiving rF-IL-2 during vaccination demonstrated higher avidity CEA-specific, as well as higher avidity gp70-specific, CD8⁺ T cells when compared with mice vaccinated without rF-IL-2. These studies demonstrate for the first time that the level and avidity of antigen specific CTL, as well as the therapeutic outcome can be improved with the use of i.t. rF-IL-2 with vaccine regimens.     

LNP-CpG ODN-adjuvanted varicella-zoster virus glycoprotein E induced comparable levels of immunity with ShingrixTM in VZV-primed mice

Latent varicella-zoster virus (VZV) may be reactivated to cause herpes zoster, which affects one in three people during their lifetime. The currently available subunit vaccine Shingrix^TM is superior to the attenuated vaccine Zostavax^® in terms of both safety and efficacy, but the supply of its key adjuvant component QS21 is limited. With ionizable lipid nanoparticles (LNPs) that were recently approved by the FDA for COVID-19 mRNA vaccines as carriers, and oligodeoxynucleotides containing CpG motifs (CpG ODNs) approved by the FDA for a subunit hepatitis B vaccine as immunostimulators, we developed a LNP vaccine encapsulating VZV-glycoprotein E (gE) and CpG ODN, and compared its immunogenicity with Shingrix^TM in C57BL/6J mice. The results showed that the LNP vaccine induced comparable levels of gE-specific IgG antibodies to Shingrix^TM as determined by enzyme-linked immunosorbent assay (ELISA). Most importantly, the LNP vaccine induced comparable levels of cell-mediated immunity (CMI) that plays decisive roles in the efficacy of zoster vaccines to Shingrix^TM in a VZV-primed mouse model that was adopted for preclinical studies of Shingrix^TM. Number of IL-2 and IFN-γ secreting splenocytes and proportion of T helper 1 (Th1) cytokine-expressing CD4⁺ T cells in LNP-CpG-adjuvanted VZV-gE vaccinated mice were similar to that of Shingrix^TM boosted mice. All of the components in this LNP vaccine can be artificially and economically synthesized in large quantities, indicating the potential of LNP-CpG-adjuvanted VZV-gE as a more cost-effective zoster vaccine.     

TcVac3 Induced Control of Trypanosoma cruzi Infection and Chronic Myocarditis in Mice

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8⁺T cell response with type-1 cytokine (IFN-γ⁺TNF-α>IL-4⁺IL-10) and cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺Perforin⁺) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn’t enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ⁺CD8⁺T cells, a predominance of immunoregulatory IL-10⁺/CD4⁺T and IL10⁺/CD8⁺T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8⁺T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.     

A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

Due to the enormous capacity of Staphylococcus aureus to acquire antibiotic resistance, it becomes imperative to develop vaccines for decreasing the risk of its life-threatening infections. Peptidoglycan (PGN) is a conserved and major component of S. aureus cell wall. However, it has not been used as a vaccine candidate since it is a thymus-independent antigen. In this study, we synthesized a multiple antigenic peptide, named MAP27, which comprised four copies of a peptide that mimics the epitope of PGN. After immunization with MAP27 five times and boosting with heat-inactivated bacterium one time, anti-MAP27 serum bound directly to S. aureus or PGN. Immunization with MAP27 decreased the bacterial burden in organs of BALB/c mice and significantly prolonged their survival time after S. aureus lethal-challenge. The percentage of IFN-γ⁺CD3⁺ T cells and IL-17⁺CD4⁺ T cells in spleen, as well as the levels of IFN-γ, IL-17A/F and CCL3 in spleen and lung, significantly increased in the MAP27-immunized mice after infection. Moreover, in vitro incubation of heat-inactivated S. aureus with splenocytes isolated from MAP27-immunized mice stimulated the production of IFN-γ and IL-17A/F. Our findings demonstrated that MAP27, as a thymus-dependent antigen, is efficient at eliciting T cell-mediated responses to protect mice from S. aureus infection. This study sheds light on a possible strategy to design vaccines against S. aureus.     

Experimental Murine Fascioliasis Derives Early Immune Suppression with Increased Levels of TGF-�� and IL-4

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19⁺ B cells was observed as early as week 1 post-infection while CD4⁺/CD8⁺ T cells were down-regulated. Accumulation of Mac1⁺ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1β expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-β were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-β and IL-4 during the early stages of infection.     

Enhanced protection against pulmonary mycobacterial challenge by chitosan‐formulated polyepitope gene vaccine is associated with increased pulmonary secretory IgA and gamma‐interferon+ T cell responses

Induction of local (pulmonary) immunity plays a critical role in preventing dissemination of Mycobacterium tuberculosis (M. tb) during the early infection stage. To induce specific mucosal immunity, chitosan, a natural cationic polysaccharide, was employed as a mucosal gene carrier and complexed with pHSP65pep, our previously constructed multi‐epitope gene vaccine, which induces splenic gamma‐interferon (IFN‐γ)⁺ T helper cell 1 responses. The resultant chitosan–pHSP65pep was administered intranasally to BALB/c mice with four doses of 50 μg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that the chitosan formulation significantly induced production of secretory immunoglobulin A (P < 0.05) as determined by measuring its concentrations in lung lavage fluid and enhanced pulmonary CD4⁺ and CD8⁺IFN‐γ⁺ T cell responses (P < 0.001) compared with naked gene vaccine. Improved protection against Mycobacterium bovis bacillus Calmette–Guérin (BCG) challenge was consistently achieved by the chitosan–DNA formulation both as the vaccine alone or in a BCG prime‐vaccine boost immunization scenario. Our study shows that mucosal delivery of gene vaccine in a chitosan formulation remarkably enhances specific SIgA concentrations and mucosal IFN‐γ⁺ T cell response, which correlated positively with immunological protection.     

Prime/Boost Immunization with DNA and Adenoviral Vectors Protects from Hepatitis D Virus (HDV) Infection after Simultaneous Infection with HDV and Woodchuck Hepatitis Virus

Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8⁺ and CD4⁺ T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven.     

Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice

Subclinical doses of Paclitaxel (PTX) given 1 day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu⁺ tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8⁺ T cells with enhanced lytic activity against neu⁺ tumors. PTX treatment also enhances maturation marker expression on CD11c⁺ DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8⁺ T cells with greater lytic capability than DC’s from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-γ-secreting CD8⁺ T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.     

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

Background

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.

Methods

HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.

Results

The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4⁺ and/or CD8⁺ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4⁺ and CD8⁺ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8⁺ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.

Conclusion

Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.

Trial Registration

International Standard Randomised Controlled Trial Number (ISRCTN) 60284968