Lack of interference with immunogenicity of a chimeric alphavirus replicon particle-based influenza vaccine by preexisting antivector immunity.

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.

Comparison of the immune responses induced by chimeric alphavirus-vectored and formalin-inactivated alum-precipitated measles vaccines in mice.

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.

A chimeric alphavirus replicon particle vaccine expressing the hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques from measles.

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-gamma)-producing T cells.

Characterization of immune responses elicited in macaques immunized sequentially with chimeric VEE/SIN alphavirus replicon particles expressing SIVGag and/or HIVEnv and with recombinant HIVgp140Env protein.

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.

Antibody responses against HIV in rhesus macaques following combinations of mucosal and systemic immunizations with chimeric alphavirus-based replicon particles.

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.

A chimeric alphavirus RNA replicon gene-based vaccine for human parainfluenza virus type 3 induces protective immunity against intranasal virus challenge.

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.

Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies.

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150(+) B95-8 cells.

Evaluation of human immunodeficiency virus type 1 subtype C gag, pol, and gagpol DNA and alphavirus replicon vaccines.

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.

Role for nsP2 proteins in the cessation of alphavirus minus-strand synthesis by host cells.

In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and approximately 10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis.

Modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles.

Measles remains a major worldwide problem partly because of difficulties with vaccination of young infants. New vaccine strategies need to be safe and to provide sustained protective immunity. We have developed Sindbis virus replicon particles that express the measles virus (MV) hemagglutinin (SIN-H) or fusion (SIN-F) proteins.

Characterization of immune responses induced by intramuscular vaccination with DNA vaccines encoding measles virus hemagglutinin and/or fusion proteins.

Measles virus (MV) hemagglutinin (MV-H) and fusion (MV-F) proteins induce plaque reduction neutralizing (PRN) antibodies and cell-mediated immune responses that protect against clinical measles. DNA vaccines that encode MV-H and MV-F are being investigated as a new generation of measles vaccine to protect infants too young to receive currently licensed attenuated measles vaccines. However, it is unclear whether DNA vaccines encoding both MV-H and MV-F act synergistically to induce stronger immunity than immunization with plasmids encoding MV-H or MV-F alone.

Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins.

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting.

Characterization of human immunodeficiency virus Gag-specific gamma interferon-expressing cells following protective mucosal immunization with alphavirus replicon particles.

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.

Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein.

We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses.

Doxorubicin and paclitaxel enhance the antitumor efficacy of vaccines directed against HER 2/neu in a murine mammary carcinoma model.

Introduction: The purpose of the present study was to determine whether cytotoxic chemotherapeutic agents administered prior to immunotherapy with gene vaccines could augment the efficacy of the vaccines.

Methods: Mice were injected in the mammary fat pad with an aggressive breast tumor cell line that expresses HER2/neu. The mice were treated 3 days later with a noncurative dose of either doxorubicin or paclitaxel, and the following day with a gene vaccine to HER2/neu.

An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector.

Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55(Gag) antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE.

Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats.

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.

Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen 85A.

The immunogenicity of a plasmid DNA vaccine incorporating Sindbis virus RNA replicase functions (pSINCP) and expressing antigen 85A (Ag85A) from Mycobacterium tuberculosis was compared with a conventional plasmid DNA vector encoding Ag85A. pSINCP-85A was highly immunogenic in mice and gave enhanced long-term protection against M. tuberculosis compared with the conventional vector.

Virus-based vectors for human vaccine applications.

Vaccinology has experienced a dramatic resurgence recently, as traditional methodologies of using attenuated live pathogens or inactivated whole pathogens have been either ineffective or are not an acceptable risk for several disease targets, including HIV and Hepatitis C. Gene-based vaccines can stimulate potent humoral and cellular immune responses, and viral vectors might be an efficient strategy for both delivery of antigen-encoding genes, as well as facilitating and enhancing antigen presentation. Vectors derived from diverse viruses with distinct tropism and gene expression strategies have been developed, and are being evaluated in preclinical and clinical vaccine studies.

Cancer immunotherapy using Sindbis virus replicon particles encoding a VP22-antigen fusion.

Alphavirus vectors have emerged as a strategy for the development of cancer vaccines and gene therapy applications. The availability of a new packaging cell line (PCL), which is capable of generating alphavirus replicon particles without contamination from replication-competent virus, has advanced the field of vaccine development. This replication-defective vaccine vector has potential advantages over naked nucleic acid vaccines, such as increased efficiency of gene delivery and large-scale production.