Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles

Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.     

Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats

Background

The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo.

Methodology/Principal Findings

The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers.

Conclusions/Significance

We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.     

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.     

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.     

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.     

Evolution of protection after maternal immunization for respiratory syncytial virus in cotton rats

Maternal anti-respiratory syncytial virus (RSV) antibodies acquired by the fetus through the placenta protect neonates from RSV disease through the first weeks of life. In the cotton rat model of RSV infections, we previously reported that immunization of dams during pregnancy with virus-like particles assembled with mutation stabilized pre-fusion F protein as well as the wild type G protein resulted in robust protection of their offspring from RSV challenge. Here we describe the durability of those protective responses in dams, the durability of protection in offspring, and the transfer of that protection to offspring of two consecutive pregnancies without a second boost immunization. We report that four weeks after birth, offspring of the first pregnancy were significantly protected from RSV replication in both lungs and nasal tissues after RSV challenge, but protection was reduced in pups at 6 weeks after birth. However, the overall protection of offspring of the second pregnancy was considerably reduced, even at four weeks of age. This drop in protection occurred even though the levels of total anti-pre-F IgG and neutralizing antibody titers in dams remained at similar, high levels before and after the second pregnancy. The results are consistent with an evolution of antibody properties in dams to populations less efficiently transferred to offspring or the less efficient transfer of antibodies in elderly dams.     

Chimeric T helper-B cell peptides induce protective response against Japanese encephalitis virus in mice

Virus neutralizing MAb binding and T helper cell stimulating peptide epitopes from structural and non-structural proteins of Japanese encephalitis virus were delineated. It was observed that priming by T helper peptides potentiated neutralizing antibody response against JE virus. Immunization with chimeric T helper - B cell peptides could thus protect mice from lethal challenge with JE virus.     

Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats

The amount of passively acquired serum respiratory syncytial virus (RSV)-neutralizing antibodies required to protect the respiratory tract of cotton rats against infection was studied. Infant cotton rats were inoculated intraperitoneally with various dilutions of a single pool of sera derived from cotton rats convalescent from RSV infection. After 24 h, these animals were inoculated with RSV intranasally. Virus replication in the respiratory tract was suppressed in cotton rats which had a serum neutralizing antibody titer of 1:100 or greater. Resistance was greater in the lungs than in the nose. Complete or almost complete resistance in the lungs was observed in cotton rats with a serum neutralizing antibody titer of 1:380 or greater. The level of serum RSV-neutralizing antibodies required to confer significant resistance to infection in the cotton rat was similar to the level of maternally derived serum antibodies possessed by human infants less than 2 months of age, who as a group exhibit relative resistance to RSV disease compared with infants 2 to 6 months of age.     

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1–3), domains I and II (D1–2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1–3, D1–2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1–3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.     

Evaluating sampling strategies for enzootic Venezuelan equine encephalitis virus vectors in Florida and Panama

Determining effective sampling methods for mosquitoes are among the first objectives in elucidating transmission cycles of vector-borne zoonotic disease, as the effectiveness of sampling methods can differ based on species, location, and physiological state. The Spissipes section of the subgenus Melanoconion of Culex represents an understudied group of mosquitoes which transmit Venezuelan equine encephalitis viruses (VEEV) in the Western Hemisphere. The objective of this study was to determine effective collection methods that target both blood-engorged and non-engorged females of the Spissipes section of Culex subgenus Melanoconion to test the hypothesis that favorable trapping methods differ between species and by physiological status within a species. Mosquitoes were collected using two commercially available traps, (CDC-light trap and BG-Sentinel trap), two novel passive traps (a novel mosquito drift fence and pop-up resting shelters), and two novel aspirators, (a small-diameter aspirator and a large-diameter aspirator) in Darién, Panama, and Florida, USA. The total number of female mosquitoes collected for each species was compared using rarefaction curves and diversity metrics. We also compared the utility of each trap for collecting total females and blood-engorged females of four Spissipes section mosquito species in Florida and Darién. In Darién, it was found that both blood-engorged and unfed females of Cx. pedroi were most effectively collected using the mosquito drift fence at 57.6% and 61.7% respectively. In contrast, the most unfed Cx. spissipes were collected using the mosquito drift fence (40.7%) while blood-engorged females were collected effectively by pop-up resting shelters (42.3%). In Florida, the best sampling technique for the collection of blood-engorged Cx. panocossa was the large diameter aspirator at 41.9%, while the best trap for collecting Cx. cedecei was the pop-up resting shelter at 45.9%. For unfed female Spissipes section mosquitoes in Florida, the CDC light trap with CO₂ collected 84.5% and 98.3% of Cx. cedecei and Cx. panocossa respectively in Florida. Rarefaction analysis, and both the Shannon and Simpsons diversity indices all demonstrated that the mosquito drift fence was capable of collecting the greatest diversity of mosquito species regardless of location. The finding that the proportions of unfed and blood-engorged mosquitoes collected by traps differed both among and between species has implications for how studies of VEEV vectors will be carried out in future investigations. In Florida a combination of pop-up resting shelters and use of a large-diameter aspirator would be optimal for the collection of both VEEV vectors for host-use studies. Results demonstrate that traps can be constructed from common materials to collect mosquitoes for VEEV vector studies and could be assessed for their utilization in vectors of other systems as well. Unfortunately, no single method was effective for capturing all species and physiological states, highlighting a particular need for assessing trap utility for target species of a study.     

A plasmid encoding Japanese encephalitis virus PrM and E proteins elicits protective immunity in suckling mice

A plasmid encoding Japanese encephalitis virus (JEV) prM and E proteins was constructed, and its efficacy as a candidate vaccine against JEV was evaluated in suckling mice. Groups of 10 BALB/c mice (5-7 days old) were immunized twice via muscular injection with this DNA vaccine, an empty vector or PBS at an interval of 3 weeks, and were challenged with a lethal dose of JEV 3 weeks after the second inoculation. Both cellular and humoral immune responses were examined before the challenge. Two animals from each group were sacrificed to detect the JEV-specific cytotoxic T lymphocyte activity. JEV-specific lactate dehydrogenase release in the DNA vaccine, empty vector and PBS groups was 37.5%, 18% and 8.5% respectively. JEV-specific antibody was detected in 8 of 10 animals in DNA vaccine group with a geometrical mean titer of 1: 28.3. The pooled serum from the same group also showed a neutralizing activity. Six of 8 mice in the DNA vaccine group survived the challenge, with a protection rate of 75%, but all the mice died in the two control groups. These results show that this JEV prM and E DNA vaccine is immunogenic and protective against JEV infection in the mouse model.     

Inability to evoke a long-lasting protective immune response to respiratory syncytial virus infection in mice correlates with ineffective nasal antibody responses

Long-lasting protective antibody is not normally generated in children following primary respiratory syncytial virus (RSV) infection, frequently leading to reinfection. We used the BALB/c mouse model to examine the role of the nasal-associated lymphoid tissue and the bone marrow in the generation of RSV-specific long-lasting plasma cells, with a view to further understanding the mechanisms responsible for the poorly sustained RSV antibody levels following primary infection. We show here that substantial numbers of RSV-specific plasma cells were generated in the bone marrow following challenge, which were maintained thereafter. In contrast, in the nasal-associated lymphoid tissue, RSV-specific plasma cell numbers waned quickly both after primary infection and after challenge and were not maintained at a higher level after boosting. These data indicate that the inability to generate a robust local mucosal response in the nasal tissues may contribute substantially to the likelihood of subsequent reinfection and that the presence of serum anti-RSV antibody without local protection is not enough to protect against reinfection.     

Congenital cerebral and ocular malformations induced in rhesus monkeys by Venezuelan equine encephalitis virus.

Rhesus monkey fetuses were inoculated with Venezuelan Equine Encephalitis (VEE) vaccine virus by the direct intracerebral route at approximately 100 days gestation to determine possible teratogenicity of the virus. Congenital micrencephaly, hydrocephalus and cataracts were found in all animals and porencephaly in 67 percent of the cases. The virus replicated in the brain and other organs of the fetus. VEE vaccine virus is teratogenic for non-human primates and must be considered a potential teratogen of man.     

Immune induction and modulation in mice following immunization with DNA encoding F protein of respiratory syncytial virus

Respiratory syncytial virus (RSV) is one of the principal agents of bronchiolitis and pneumonia in young children. Thus, there is a strong need to make a safe and effective vaccine against the RSV infection. DNA immunization is very effective at inducing both cellular and humoral immune responses. In this study, we inserted the RSV-F gene into expression vectors, pcDNA3.1 and pQE. These constructs were transformed into C2C12 and E. coli M15 cells, respectively. The expression of the RSV-F protein was confirmed by SDS-PAGE, followed by Western blot analyses. The immunization of pcDNA3.1-RSV-F elicited both anti-RSV-F titer in mouse sera and CTL activities with mouse splenocytes. Especially, the co-administration of IL-4, or the GM-CSF gene with the RSV-F gene construct, enhanced the production of anti-RSV-F Ab. However, this enhancement disappeared by the simultaneous injection of the Th1 and Th2 type cytokine genes. The CTL activities were affected by the co-delivery of the IFN-gamma gene, but not by Th2-type cytokines.     

Mechanism of antibody-mediated viral clearance in immunotherapy of respiratory syncytial virus infection of cotton rats.

Antibody-mediated clearance of respiratory syncytial virus from cotton rat pulmonary tissues occurs in the absence of complement and in the absence of the Fc portion of the immunoglobulin G molecule, suggesting that complement-independent, cell-independent neutralization is the major mechanism of clearance.     

Efficacy and safety studies of a recombinant chimeric respiratory syncytial virus FG glycoprotein vaccine in cotton rats

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.     

Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector.

A molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE) has been genetically configured as a replication-competent vaccine vector for the expression of heterologous viral proteins (N. L. Davis, K. W. Brown, and R. E. Johnston, J. Virol. 70:3781-3787, 1996). The matrix/capsid (MA/CA) coding domain of human immunodeficiency virus type 1 (HIV-1) was cloned into the VEE vector to determine the ability of a VEE vector to stimulate an anti-HIV immune response in mice. The VEE-MA/CA vector replicated rapidly in the cytoplasm of baby hamster kidney (BHK) cells and expressed large quantities of antigenically identifiable MA/CA protein. When injected subcutaneously into BALB/c mice, the vector invaded and replicated in the draining lymphoid tissues, expressing HIV-1 MA/CA at a site of potent immune activity. Anti-MA/CA immunoglobulin G (IgG) and IgA antibodies were present in serum of all immunized mice, and titers increased after a second booster inoculation. IgA antibodies specific for MA/CA were detected in vaginal washes of mice that received two subcutaneous immunizations. Cytotoxic T-lymphocyte responses specific for MA/CA were detected following immunization with the MA/CA-expressing VEE vector. These findings demonstrate the ability of a VEE-based vaccine vector system to stimulate a comprehensive humoral and cellular immune response. The multifaceted nature of this response makes VEE an attractive vaccine for immunization against virus infections such as HIV-1, for which the correlates of protective immunity remain unclear, but may include multiple components of the immune system.     

Enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactiva ted virus.

Cotton rats previously inoculated with Formalin-inactivated respiratory syncytial virus (RSV) were challenged intranasally with live RSV to induce an enhancement of RSV disease similar to that observed after the administration of Formalin-inactivated RSV vaccine to human infants 20 years ago. Within 24 h after infection with RSV, cotton rats developed pulmonary lesions that reached a maximum by day 4. Histologically, the lesions resembled an experimental pulmonary Arthus reaction. An action of Formalin on RSV appears to be responsible for this effect, because live virus or virus heated in the absence of Formalin did not induce enhanced immunopathology. Selected epitopes on the fusion (F) or attachment (G) or both RSV surface glycoproteins that are involved in inducing neutralizing antibodies were modified to reduce or ablate their antigenicity. However, other epitopes on the F or G or both glycoproteins were not ablated by Formalin, because cotton rats inoculated parenterally with a Formalin-inactivated virus developed a high level of F and G antibodies measurable by an enzyme-linked immunosorbent assay. At this time, the effect of Formalin on RSV cannot be localized to either the F or G glycoprotein of RSV.