ABSTRACT Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens.

ABSTRACT Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-γ)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-γ-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.

A variety of vaccine platforms are under study for development of new vaccines for measles. Problems with past measles vaccines are incompletely understood and underscore the need to understand the types of immune responses induced by different types of vaccines. Detailed immune response evaluation is most easily performed in mice. Although mice are not susceptible to infection with wild type or vaccine strains of measles virus, they can be used for comparative evaluation of the immune responses to measles vaccines of other types. In this study we compared the immune responses in mice to a new protective alphavirus replicon particle vaccine expressing the measles virus hemagglutinin (VEE/SIN-H) with a non-protective formalin-inactivated, alum-precipitated measles vaccine (FI-MV). MV-specific IgG levels were similar, but VEE/SIN-H antibody was high avidity IgG2a with neutralizing activity while FI-MV antibody was low-avidity IgG1 without neutralizing activity. FI-MV antibody was primarily against the nucleoprotein with no priming to H. Germinal centers appeared, peaked and resolved later for FI-MV. Lymph node MV antibody-secreting cells were more numerous after FI-MV than VEE/SIN-H, but were similar in the bone marrow. VEE/SIN-H-induced T cells produced IFN-gamma and IL-4 both spontaneously ex vivo and after stimulation, while FI-MV-induced T cells produced IL-4 only after stimulation. In summary, VEE/SIN-H induced a balanced T cell response and high avidity neutralizing IgG2a while FI-MV induced a type 2 T cell response, abundant plasmablasts, late germinal centers and low avidity non-neutralizing IgG1 against the nucleoprotein.

Vaccination strategies that can block or limit heterosexual human immunodeficiency virus (HIV) transmissions to local and systemic tissues are the goal of much research effort. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein-based vaccines, applies to immunizations with DNA- or RNA-based vectors. Intranasal (i.n.) followed by intramuscular (i.m.) immunizations (i.n./i.m.) with polylactide-coglycolide (PLG)-DNA microparticles encoding HIV-gag (PLG-DNA-gag) significantly enhanced serum antibody responses, compared with i.m., i.n. or i.m. followed by i.n. (i.m./i.n.) immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG-DNA-gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, beta 7-deficient mice developed local and systemic antibody responses following i.n./i.m. immunization, or immunization via any other route, similar to those of wild-type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN-gag replicon particles, composed of Venezuelan equine encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., compared with any other immunizations, i.n./i.m. immunization with VEE/SIN-gag resulted in enhanced genital tract but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B-cell responses independent of the mucosal homing receptors alpha 4 beta 7 and alpha E beta 7.

Parainfluenza virus type 3 (PIV3) infections continue to be a significant health risk for infants, young children, and immunocompromised adults. We describe a gene-based vaccine strategy against PIV3 using replication-defective alphavirus vectors. These RNA replicon vectors, delivered as virus-like particles and expressing the PIV3 hemagglutinin-neuraminidase glycoprotein, were shown to be highly immunogenic in mice and hamsters, inducing PIV3-specific neutralizing antibody responses. Importantly, the replicon particle-based vaccine administered intramuscularly or intranasally protected against mucosal PIV3 challenge in hamsters, preventing virus replication in both nasal turbinates and lungs. These data suggest that the alphavirus replicon platform can be useful for a PIV3 vaccine and possibly other respiratory viruses.

Mucosal and systemic transmission of HIV is prevalent. Therefore, mucosal followed by parenteral immunizations with chimeric vs. complete alphavirus-based replicon particles, encoding an HIV envelope glycoprotein, were tested. Female rhesus macaques were immunized intranasally and then intramuscularly. Following the immunizations, enhanced mucosal and systemic antibody responses were detected with the chimeric compared to the complete replicon particles. Although similar proportions of the same peripheral blood monocyte lineage target cells were infected with the chimeric vs. the complete replicon particles, the latter resulted in enhanced expression of the gene of interest, suggesting a possible mechanism of the enhanced immunogenicity.

The primary objective of our current HIV vaccine research program is to evaluate novel alphavirus-based and Envelope (Env) protein-based vaccine formulations either alone or in prime boost regimen for improved immunogenicity and efficacy over other HIV vaccine approaches. The outcome of this work will be the production of candidate vaccine components that can be further advanced to clinical trials in human subjects. For these studies we employ viral genes derived from SF162 a primary subtype B HIV-1 isolate in alphavirus replicon particle-based vaccines. A chimeric alphavirus vector system (VEE/SIN) is used that is composed of the Venezuelan Equine Encephalitis (VEE) replicon combined with the Sindbis virus structural proteins. We have performed a proof of concept study where macaques were primed with VEE/SIN, boosted with o-gp140DV2SF162, and challenged intravenously by pathogenic SHIVSF162P4. In the next study we evaluated these recombinant VEE/SIN vaccines alone, and also in combination with DV2-trimer derived from SF162 for their ability to protect against mucosal SHIVSF162P4 challenge. Both parenteral and mucosal routes of immunization have been evaluated to identify the optimal regimen(s) for the induction of potent and protective humoral and cellular responses in rhesus macaques. In the third study, we evaluated the ability of DV2SF162 trimer to protect against against mucosal SHIVSF162P4 challenge. Both parenteral and mucosal routes of immunization have been evaluated. Results so far indicate that the recombinant VEE/SIN particles induced antigen-specific CD4+ and CD8+ T cell responses as well as neutralizing antibodies in small animals (mice and rabbits) and non-human primates. Moreover, intramuscular immunization with VEE/SIN particles encoding SIV gag or gagpol and HIV env is quite effective in priming for potent anti-Env antibody responses. However, it seems intramuscular boosting of alpha primed animals with o-gp140DV2SF162 in MF59 is critical for blocking and/or reducing acute phase viremia following either intravenous or intrarectal SHIVSF162P4 challenges of rhesus macaques. Furthermore, macaques that were immunized intranasally with o-gp140DV2SF162 protein in the LTK63 mucosal adjuvant followed by intramuscular immunization with o-gp140DV2SF162 in MF59 were also protected against SHIVSF162P4 challenge. Protection in these studies appears to occur after the induction of high titer serum neutralizing antibodies that recognize the challenge virus.

In the present study, macaques were coimmunized with VEErep/SINenv chimeric alphavirus replicon particles expressing SIVp55Gag and HIVDeltaV2gp140Env or only with replicon particles expressing HIVDeltaV2gp140Env. All animals were subsequently immunized with recombinant trimeric HIVDeltaV2gp140Env protein. During alphavirus immunization, anti-SIVGag and anti-HIVEnv-specific interferon (IFN)-gamma responses, as well as high titers of anti-HIVEnv binding (gp120 but not gp41 specific) and anti-HIV neutralizing antibodies, were generated. The subsequent immunization with recombinant HIVDeltaV2gp140 enhanced the neutralizing antibody titers and Env-specific IFN-gamma responses. Following intravenous challenge with the R5- tropic SHIV(SF162P4) virus, significantly lower primary plasma viremia levels were recorded in the immunized animals, as compared to control animals immunized with replicon particles expressing influenza virus HA. Our results show that this method of immunization elicits both strong cellular immunity and neutralizing antibodies in primates and, thus, merits further investigation.

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8+ T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press [1]) and support a subtype C in-frame gagpol fusion gene vaccine.

ABSTRACT A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8 + intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed α4β7 or α Eβ 7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed α4β7 or CD62L than expressed α Eβ 7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.

Objectives: To evaluate the immunogenicity of sequence-modified HIV env and gag in baboons using DNA prime and protein boost strategy. Methods: Synthetic sequence-modified HIV gene cassettes were constructed that expressed three different forms of Env proteins, gp140, gp140mut and gp140TM, plus or minus a mutation in the protease-cleavage site. These plasmids were used to immunize baboons (Papio cynocephalus). A group of baboons was also immunized with both env and gag DNA followed by p55Gag virus-like particles (VLP) boost. Results: Modest antibody responses and low or no lymphoproliferative responses were observed following multiple DNA immunizations. In contrast, strong antibodies and substantial antigen-specific lymphoproliferative responses were seen following booster immunizations with oligomeric Env protein (o-gp140US4) in MF59. Neutralizing antibody responses were scored against T cell line adapted HIV-1 strains after the protein boosters, but neutralizing responses were low or absent against homologous and heterologous primary isolate strains. In the group receiving both gag and env vaccines, modest antigen-specific antibody and lymphoproliferative responses were scored after the DNA immunizations; these responses were enhanced several-fold upon boosting with the VLP preparations. The addition of Gag antigen did not interfere with Env-specific antibody responses, but there was a negative effect on the levels of Env-specific lymphoproliferation. Conclusions: These results highlight the importance of improving the potency of HIV DNA vaccines by enhanced DNA delivery and prime-boost vaccine technologies to generate more robust immune responses in larger animal models. In addition, care must be taken when immunizations with Env and Gag antigens are performed together.

Several vaccine technologies were evaluated for their abilities to induce anti-human immunodeficiency virus Gag immune responses in rhesus macaques. While no vaccine alone was able to induce broad and strong immune responses, these were achieved by priming with Gag DNA and boosting with Gag protein adsorbed to polylactide coglycolide microparticles. This regimen elicited strong antibodies, helper T cells, and cytotoxic T lymphocytes and thus holds promise as an effective vaccination scheme.

The majority of human immunodeficiency virus (HIV) infections occur through vaginal and rectal transmission. In seeking a safe, nonreplicating gene-delivery vector that can induce mucosal and systemic immune responses and protection, Sindbis virus-based replicon particles expressing HIV-1 Gag (SIN-Gag) were developed. In mice, after nasal or intramuscular immunization with SIN-Gag and vaginal challenge with vaccinia virus (VV) expressing HIV-1 Gag (VV-Gag), CD8(+) T cell-mediated responses were detected locally, in the vaginal mucosa and in the draining iliac lymph nodes (ILNs), and systemically, in the spleen. However, the mice were not protected against VV-Gag replication in the ovaries. In contrast, after vaginal or rectal immunization with SIN-Gag and vaginal challenge with VV-Gag, despite lower local CD8(+) T cell-mediated responses in the vaginal mucosa and ILNs, the mice were protected against VV-Gag replication in the ovaries. Therefore, local immunization with SIN-Gag induced both local mucosal cell-mediated responses and protection.