Alphavirus Replicon Research Articles

Alphavirus replicon particles acting as adjuvants promote CD8 + T cell responses to co-delivered antigen

Alphavirus replicon particles induce strong antibody and CD8 + T cell responses to expressed antigens in numerous experimental systems. We have recently demonstrated that Venezuelan equine encephalitis virus replicon particles (VRP) possess adjuvant activity for systemic and mucosal antibody responses. In this report, we demonstrate that VRP induced an increased and balanced serum IgG subtype response to co-delivered antigen, with simultaneous induction of antigen-specific IgG1 and IgG2a antibodies, and increased both systemic and mucosal antigen-specific CD8 + T cell responses, as measured by an IFN-γ ELISPOT assay. Additionally, VRP further increased antigen-specific T cell immunity in an additive fashion following co-delivery with the TLR ligand, CpG DNA. VRP infection led to recruitment of CD8 + T cells into the mucosal compartment, possibly utilizing the mucosal homing receptor, as this integrin was upregulated on CD8 + T cells in the draining lymph node of VRP-infected animals, where VRP-infected dendritic cells reside. This newly recognized ability of VRP to mediate increased T cell response towards co-delivered antigen provides the potential to both define the molecular basis of alphavirus-induced immunity, and improve alphavirus-based vaccines.

The contribution of type I interferon signaling to immunity induced by alphavirus replicon vaccines

The contribution of type I interferon signaling to immunity induced by alphavirus replicon vaccines

Alphavirus replicon particles acting as adjuvants promote CD8+ T cell responses to co-delivered antigen

Alphavirus replicon particles acting as adjuvants promote CD8+ T cell responses to co-delivered antigen

Advances in the development of vaccines against Marburg and Ebola viruses.

Peter L Collins & Alexander Bukreyev† †Author for correspondence Laboratory of Infectious Disease, National Institute of Allergy & Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6505, Bethesda, MA 20892-8007, USA Tel.: +1 301 594 1854; Fax: +1 301 496 8312; ab176v@nih.gov ‘The development of vaccines against MARV and EBOV ... is a high priority because of their high virulence, ability to spread from person to person and the possibility of their use in bioterrorism.’

Development and preclinical evaluation of an alphavirus replicon vaccine for influenza

We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-γ ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.

Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus

We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-γ ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.

Alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in BALB/c mice and functional serum antibodies in rhesus macaques

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-γ + splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.

Alphavirus derived vector, still a relevant system through new mobilisation procedure

Among eukaryotic cell-expression-systems, the one derived from alphaviruses, including Semliki forest virus (SFV), offers an efficient method for protein production in mammalian cells. Despite this efficacy, twenty years after their discovery alphaviruses vectors remain poorly used. Alphavirus vectors exist as naked RNA vectors or as recombinant particles. The use of costly RNA-based replicons, and the fact that production of recombinant particles is a complex process to carry out, have hampered the attractiveness of the methods. Lastly, the apoptotic signals induced by alphavirus vectors replication leads to a rapid death of the producing cells. This feature, which can be detrimental in vitro, is advantageously exploited for in vivo applications. Besides laboratory applications, alphavirus vectors have been explored in rare phase I clinical trials, for vaccine development and cancer gene therapy, therefore, alphavirus vector will benefit from the advent of new, biosafety-efficient, methods for particles production. Most of the recent advances in the field proposed an heterologous mobilisation of alphavirus replicon. While increasing biosafety aspects, new methods are also simpler regarding the genesis of recombinant particles. In the present review, we overview the alphavirus life cycle with a special attention to the features influencing vector design and utility.

Protection of pigs from lethal challenge by a DNA vaccine based on an alphavirus replicon expressing the E2 glycoprotein of classical swine fever virus

In a previous study, it has been shown that a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) conferred full protection for pigs immunized three times with 600 μg of the vaccine. This study was designed to evaluate further the efficacy of the vaccine with lower dosage and fewer inoculations. Pigs were immunized twice with 100 μg of pSFV1CS-E2 ( n = 5) or control plasmid pSFV1CS ( n = 3), respectively, and challenged with virulent Shimen strain 6 weeks following the booster immunization. Pigs immunized with pSFV1CS-E2 developed high titers of specific neutralizing antibodies against CSFV after the booster, and the antibody titers increased rapidly upon challenge. The immunized animals showed no clinical symptoms except short-term fever and low-level viremia, whereas, the control pigs immunized with the control plasmid produced no detectable antibody prior to challenge, and showed obvious clinical signs following challenge, and two pigs died of illness. All control animals developed extended viremia as detected by nested RT-PCR and real-time RT-PCR. Severe pathologic lesions typical of CSFV infection were observed at necropsy. It is concluded that the alphavirus replicon-vectored DNA-based vaccine can be a potential marker vaccine against CSFV.

Cellular and Humoral Immune Responses to Alphavirus Replicon Vaccines Expressing Cytomegalovirus pp65, IE1, and gB Proteins

Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusion protein, or the extracellular domain of gB and with double-promoter replicons expressing pp65 and IE1. Protein expression was lower with double- and triple-promoter replicons expressing gB, especially the full-length form of gB. BALB/c mice immunized with VRP expressing gB developed high titers of neutralizing antibody to CMV, and mice immunized with VRP expressing pp65, IE1, or a pp65/IE1 fusion protein developed robust antigen-specific T-cell responses as measured by gamma interferon enzyme-linked immunospot assay. Three overlapping immunodominant pp65 peptides contained a nine-amino-acid sequence (LGPISGHVL) that matches the consensus binding motif for a major histocompatibility complex H2-D(d) T-cell epitope. These data provide the basis for further development and clinical evaluation of an alphavirus replicon vaccine for CMV expressing the pp65, IE1, and gB proteins.

Evaluation of cellular immune responses induced in mice by alphavirus replicon particle vaccines for SARS-CoV (B190)

Abstract An RNA replicon vector system derived from an attenuated strain of VEE virus was used to produce virus-like replicon particles (VRP) expressing SARS-CoV genes. We previously showed in mouse studies that complete protection was conferred by SARS S VRP and partial protection with SARS M VRP. In this study, two assays for cellular immunity were used to characterize the T cell phenotypes and cytokine profiles induced after SARS VRP vaccination. Following SARS M, N, S or E VRP immunization of BALB/c mice, splenocytes were harvested for antigen specific ICS and ELISPOTs. The SARS S VRP induced both CD4 and CD8 responses with the latter being the dominant component as measured by ICS for IFN-γ or TNF-α. Polyfunctional CD8 cells secreting both IFN-γ and TNF-α were also detected. Following SARS N VRP immunization, T cells secreting IFN-γ or TNF-α were readily detected with cells secreting both predominately identified as CD4 cells. Th1 cytokine secretion was not detected in cells from SARS M VRP inoculated mice. SARS E VRP elicited a robust CD8 response as shown by cytokine expression in cells stimulated with a 9mer peptide. IL-4 was not detectable after any SARS VRP vaccination. The IFN-γ ELISPOT results paralleled those from ICS, indicating that the SARS VRP vaccines induced Th1 biased responses, although the cytokines elicited differed depending on the SARS gene expressed. This study was supported by NIH grant #UC1-AI62582

Evaluation of the immunogenicity of an alphavirus replicon particle vaccine expressing the SARS-CoV spike (S) glycoprotein in non-human primates (B191)

Abstract We previously evaluated the immunogenicity and efficacy of a virus-like replicon particle (VRP) vaccine expressing the SARS CoV S glycoprotein in mice. SARS S VRP were highly immunogenic and conferred protection upon challenge. To extend that work we have evaluated the immune responses induced by SARS S VRP in macaques immunized at 0, 4 and 24 weeks. PBMC were assayed at two week intervals by ELISPOT and ICS. Post prime, both CD4 and CD8 T cells were found that secrete IFN- γ, IL-2 and TNF- α. The percentage of CD4 cells secreting IFN- γ, IL-2 and TNF- α was substantially more than the percentage of CD4 cells secreting IL-4 post prime indicating a Th1 bias. Post boost, the number of CD4 cells secreting all measured cytokines were equivalent indicating that the SARS S VRP induced a balanced Th1/Th2 response after 2 inoculations. By week 8 a measured decline in the percentage of cytokine-secreting CD8 cells was observed. There were no significant differences in the numbers of T cells secreting cytokines with 2 different VRP dosages given as measured by ICS, however a dose response was seen by ELISPOT. Neutralizing antibodies (NAb) to SARS CoV were measured in sera following post prime and boost inoculations. Three of 4 animals in the low dose group had measurable NAb post boost (GMT = 101) whereas all 4 animals in the high dose group had serum NAb after two doses (GMT = 190). These data provide support to further development of a SARS S VRP vaccine. This study was supported by NIH grant #UC1-AI62582

Preconceptual Administration of an Alphavirus Replicon UL83 (pp65 Homolog) Vaccine Induces Humoral and Cellular Immunity and Improves Pregnancy Outcome in the Guinea Pig Model of Congenital Cytomegalovirus Infection

Development of a vaccine against congenital cytomegalovirus (CMV) infection is a major public health priority. We report the use of a propagation-defective, single-cycle, RNA replicon vector system, derived from an attenuated strain of the alphavirus Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRPs) expressing GP83, the guinea pig CMV (GPCMV) homolog of the human CMV pp65 phosphoprotein. Vaccination with VRP-GP83 induced antibodies and CD4(+) and CD8(+) T cell responses in GPCMV-seronegative female guinea pigs. Guinea pigs immunized with VRP-GP83 vaccine or with a VRP vaccine expressing influenza hemagglutinin (VRP-HA) were bred for pregnancy and subsequent GPCMV challenge during the early third trimester. Dams vaccinated with VRP-GP83 had improved pregnancy outcomes, compared with dams vaccinated with the VRP-HA control. For VRP-GP83-vaccinated dams, there were 28 live pups and 4 dead pups (13% mortality) among 10 evaluable litters, compared with 9 live pups and 12 dead pups (57% mortality) among 8 evaluable litters in the VRP-HA-vaccinated group (P<.001, Fisher's exact test). Improved pregnancy outcome was accompanied by reductions in maternal blood viral load, measured by real-time polymerase chain reaction. These results indicate that cell-mediated immune responses directed against a CMV matrix protein can protect against congenital CMV infection and disease.

Recombination-ready Sindbis replicon expression vectors for transgene expression

BackgroundSindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation.ResultsRecombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis.ConclusionReplication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

Alphavirus replicon approach to promoterless analysis of IRES elements

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced > 95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

Antibody mediated protection of immunized macaques against pathogenic SHIV challenge

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.

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. 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.

Development and characterization of a Rift Valley fever virus cell–cell fusion assay using alphavirus replicon vectors

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell–cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by β-galactosidase α-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell–cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell–cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

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. 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 adjuvant activity of alphavirus replicon particles

Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines.