Live Vaccine Vector Research Articles

A New Generation of Stable, Nonantibiotic, Low-Copy-Number Plasmids Improves Immune Responses to Foreign Antigens in Salmonella enterica Serovar Typhi Live Vectors

We hypothesized that adequately engineered attenuated Salmonella enterica serovar Typhi strains can serve as multivalent mucosal live vector vaccines to immunize against unrelated human pathogens. Toward this ultimate goal, we have developed a novel genetic stabilization system for antigen-expressing plasmids, engineered to encode the single-stranded binding protein (SSB), an essential protein involved in DNA metabolism which was deleted from the live vector chromosome. We utilized full-length protective antigen (PA83) of anthrax toxin from Bacillus anthracis as a foreign antigen and expressed PA83 as a fusion with the ClyA export protein, which allows export of ClyA-PA83 to the surface of S. Typhi live vectors. A series of SSB-encoding multicopy expression plasmids were introduced into reengineered S. Typhi strains previously tested in clinical trials, i.e., CVD 908-htrA and its less attenuated parent CVD 908. Immunogenicity was examined using a mouse model of intranasal immunization with live vector, followed by parenteral boosting with purified PA83. PA-specific antibody responses markedly improved as the copy number of the SSB-encoding plasmids decreased, and this effect was dramatically enhanced when the foreign antigen was delivered by the less attenuated live vector CVD 908ssb. These results suggest that antibody responses to antigens delivered by S. Typhi live vectors are inversely related to the metabolic burden imposed by expression of the foreign antigen and that these responses can be improved when antigens are expressed from low-copy-number plasmids and exported out of the cytoplasm of less attenuated live vectors.

Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD 103-HgR.

Live, attenuated Vibrio cholerae vaccines can induce potent immune responses after only a single oral dose. The strategy of harnessing these strains to present antigens from heterologous pathogens to the mucosal immune system shows great promise. To fully realize this possibility, V. cholerae strains must be created that stably express antigens in vivo in sufficient quantity to generate an immune response. In vivo-induced promoters have been shown to increase the stability and immunogenicity of foreign antigens expressed from multicopy plasmids. We report the construction of a series of genetically stabilized plasmids expressing luciferase as a heterologous protein from the following in vivo-induced promoters: V. cholerae P(argC), P(fhuC) and P(vca1008), and Salmonella enterica serovar Typhi P(ompC). We demonstrate that several of these expression plasmids meet two critical criteria for V. cholerae live vector vaccine studies. First, the plasmids are highly stable in the V. cholerae vaccine strain CVD 103-HgR at low copy number, in the absence of selective pressure. Second, real-time bioluminescent imaging (BLI) demonstrates inducible in vivo expression of the promoters in the suckling mouse model of V. cholerae colonization. Moreover, the use of BLI allows for direct quantitative comparison of in vivo expression from four different promoters at various time points.

A prime–boost immunisation regimen using recombinant BCG and Pr55gag virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55gag virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55gag VLPs.

Development of prophylactic recombinant HPV58-attenuated <italic>Shigella</italic> live vector vaccine and evaluation of its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model

To develop a prophylactic recombinant HPV58L1-attenuated Shigella live vector vaccine and evaluate its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model, the HPV58L1 gene was cloned into vector pUCmt, and then subcloned into the suicide vector pCVD442. The recombinant plasmid pCVD442-HPV58L1 was introduced into attenuated Shigella (sf301:deltavirG) with the helper plasmid PRK2013 by filter mating. The positive colonies were harvested and confirmed by polymerase chain reaction. The expression of the HPV58L1 protein with a molecular weight of 60 kDa was confirmed by western blot. The ability of the interested protein to self-assemble into virus-like particles was identified by transmission electron microscope, and murine erythrocyte hemagglutination assay. The guinea pig keratoconjunctivitis model was used to evaluate the protective efficacy and immunogenicity of the vaccine. Animal experiments showed that there was no keratoconjunctivitis occurred in the immunized group (HPV58-attenuated Shigella), and the serum levels of anti-HPV58L1-IgG and -IgA were obviously increased (P < 0.05), but the anti-sf301 LPS-IgG just slightly increased (P > 0.05). Enzymelinked immunosorbent spot assay showed that HPV58L1-specific IgA-antibody-secreting cells (ASC) and IgG-ASC of spleen and lymph nodes were also obviously increased (P < 0.01). In this study, a recombinant HPV58L1-attenuated Shigella live vector vaccine was successfully constructed, and it could induce strong humoral immune responses in the immunized animals, and induce protective antibody production.

Advances in fowlpox vaccination.

Abstract Fowlpox virus (FWPV) is widespread, causing disease of mature poultry with significant social or economic impact and some mortality, which is higher in the diphtheritic form of the disease. Vaccines against the disease were some of the earliest developed for livestock. They are live vaccines based on attenuated isolates or passaged derivatives of FWPV, or the serologically closely related pigeonpox virus. Relying heavily on approaches developed for vaccinia virus, FWPV was developed as a live recombinant vaccine vector for use in poultry. Licensed, commercial, recombinant FWPV vaccines are available against avian influenza, Newcastle disease and infectious laryngotracheitis. Causing disease only in birds, FWPV is nevertheless able to infect mammalian cells, express proteins and induce immune responses, both humoral and cellular, which may in some circumstances be protective (a feature shared with another member of the genus, canarypox virus). Blocked during morphogenesis (or before) in mammalian cells, it has an extremely high safety profile as a live recombinant vaccine vector for use in humans and other mammals, as demonstrated in numerous clinical trials. Relatively low potency in mammals has been addressed by using prime-boost regimes and by co-expression of host cytokines (an approach also trialled in poultry) or co-stimulators. New generation vectors may involve deletion of FWPV immunomodulators to improve efficacy.

Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease

Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1–3 × 10 8 TCID 50 Ad-P12 × 3C recombinant adenovirus were completely protected against 10,000 GID 50 homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12 × 3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1 × 10 9 TCID 50 Ad-P12 × 3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID 50) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12 × 3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

What Are the Risks—Hypothetical and Observed—of Recombination Involving Live Vaccines and Vaccine Vectors Based on Nonsegmented Negative-Strain RNA Viruses?

Newcastle disease virus (NDV) is a nonsegmented negative-strand RNA virus (NNSV) that is being developed as a potential vaccine vector for use in poultry (7, 12, 17) and humans (5, 6). The primary proposed human use would be to express protective antigens of highly pathogenic agents for outbreak control. We noted (1) that one of the advantages of NDV is that “gene exchange seems to be rare for nonsegmented negative strand RNA viruses, with few reported instances. This differs to the frequent gene reassortment observed for segmented viruses, such as influenza virus and rotavirus, and the high frequency of recombination observed for certain viruses, such as coronavirus and poliovirus.” In a recent letter to the editor (9), Han et al. criticized our studies, suggesting that we (i) dismissed the possibility of recombination, (ii) failed to recognize the potentially adverse consequences of recombination, and (iii) did not experimentally address the potential for the instability of the inserted foreign gene.

A Replication-Limited Recombinant Mycobacterium bovis BCG Vaccine against Tuberculosis Designed for Human Immunodeficiency Virus-Positive Persons Is Safer and More Efficacious than BCG

Tuberculosis is the leading cause of death in AIDS patients, yet the current tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is contraindicated for immunocompromised individuals, including human immunodeficiency virus-positive persons, because it can cause disseminated disease; moreover, its efficacy is suboptimal. To address these problems, we have engineered BCG mutants that grow normally in vitro in the presence of a supplement, are preloadable with supplement to allow limited growth in vivo, and express the highly immunoprotective Mycobacterium tuberculosis 30-kDa major secretory protein. The limited replication in vivo renders these vaccines safer than BCG in SCID mice yet is sufficient to induce potent cell-mediated and protective immunity in the outbred guinea pig model of pulmonary tuberculosis. In the case of one vaccine, rBCG(mbtB)30, protection was superior to that with BCG (0.3-log fewer CFU of M. tuberculosis in the lung [P < 0.04] and 0.6-log fewer CFU in the spleen [P = 0.001] in aerosol-challenged animals [means for three experiments]); hence, rBCG(mbtB)30 is the first live mycobacterial vaccine that is both more attenuated than BCG in the SCID mouse and more potent than BCG in the guinea pig. Our study demonstrates the feasibility of developing safer and more potent vaccines against tuberculosis. The novel approach of engineering a replication-limited vaccine expressing a recombinant immunoprotective antigen and preloading it with a required nutrient, such as iron, that is capable of being stored should be generally applicable to other live vaccine vectors targeting intracellular pathogens.

Lactococcus lactis-expressing listeriolysin O (LLO) provides protection and specific CD8+ T cells against Listeria monocytogenes in the murine infection model

Lactococcus lactis has previously been proposed as a vaccine platform for the safe delivery of heterologous antigens. Here we utilized L. lactis as a live vector for expression of listeriolysin O (LLO), a major Listeria monocytogenes antigen and virulence factor. A variety of plasmid constructs were designed to permit either constitutive or nisin-inducible expression of secreted or non-secreted LLO in L. lactis. Recombinant strains were subsequently tested in a murine model for vaccination efficacy against L. monocytogenes infection. CD8+ T lymphocytes specific for the LLO91–99 epitope were detected when strains were administered via the intraperitoneal (IP) but not the oral route. Challenge with live L. monocytogenes revealed different levels of protection among the three vaccine strains tested with the nisin-inducible LLO-secreting L. lactis strain providing the greatest protection against secondary infection. This work highlights the usefulness of the GRAS (Generally Regarded As Safe) organism L. lactis as the basis of a live vaccine vector against L. monocytogenes. The work suggests that LLO-expressing L. lactis strains may also have the potential to act as a platform for directing other co-expressed antigens towards the cytosolic MHC class I pathway for enhanced stimulation of the CD8+ T-cell response.

Emerging vaccines for influenza

Background: Influenza remains one of the leading causes of morbidity and mortality worldwide. The available vaccines are least effective in the populations at greatest risk – children, the elderly, and the immunocompromised. Furthermore, avian influenza and other novel strains have the potential to cause the next influenza pandemic. Research efforts have accelerated worldwide to develop new vaccines to provide better immunity against annual epidemics and a potential pandemic. Objective: To summarize the global research efforts at developing new influenza vaccines, adjuvants, and delivery devices. Method: MEDLINE and Pharmaprojects databases were searched for publications and continuing research on new influenza vaccine technologies. Results/conclusions: Technologies such as DNA vaccines, live recombinant viral vector vaccines, and virus-like particles have shown significant promise for immunogenicity and protection from experimental challenge to influenza. New modalities for vaccine delivery and methods for rapid vaccine production are also being investigated. With the possibility of an influenza pandemic increasing the need to develop new vaccines, the global research community has made large strides to meet this challenge.

Mise au point d’un vaccin prophylactique contre l’infection par le VIH. Où en est la recherche clinique?

Purpose The human immunodeficiency virus (HIV) and the acquired immunodeficiency syndrome induce account for over 40 million deaths in the past 20 years. Given that the currently available treatments to prevent HIV transmission and disease are not effective in eradicating the virus, vaccination likely represents the only efficacious adapted response to the global impact of this infection. This paper reviews the challenges encountered in the development of an HIV vaccine as well as the different vaccine approaches and main HIV vaccine candidates evaluated in clinical trials. Current knowledge and key points In spite the tremendous progress in HIV research, the major challenges that are encountered in the development of an HIV vaccine remain of scientific order and include viral specificities, absence of correlates of immune protection and limitations of existing animal models. Over 30 vaccine candidates have been evaluated in clinical trials. These vaccine approaches include the use of recombinant envelope proteins, DNA vaccines, live-vectored recombinant vaccines, subunit vaccines and prime-boost regimens combining various vaccine candidates. Although the protective efficacy of these candidate vaccines has yet to be demonstrated, some vaccination regiments appear to dampen initial viremia and prolong disease-free survival. Future prospects and projects Faced with the challenges in developing an HIV vaccine, international consortia and new methodologies have been proposed in order to accelerate the development and screening process of new candidate HIV vaccines. Moreover, in the absence of a protective vaccine, the impact of a vaccine that confers partial protection needs to be seriously considered.

Type IVB Pilus Operon Promoter Controlling Expression of the Severe Acute Respiratory Syndrome-Associated Coronavirus Nucleocapsid Gene in Salmonella enterica Serovar Typhi Elicits Full Immune Response by Intranasal Vaccination

Attenuated Salmonella enterica serovar Typhi strains have been considered to be attractive as potential live oral delivery vector vaccines because of their ability to elicit the full array of immune responses in humans. In this study, we constructed an attenuated S. enterica serovar Typhi strain stably expressing conserved nucleocapsid (N) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) by integrating the N gene into the pilV gene, which was under the control of the type IVB pilus operon promoter in S. enterica serovar Typhi. BALB/c mice were immunized with this recombinant strain through different routes: intranasally, orogastrically, intraperitoneally, and intravenously. Results showed that the intranasal route caused the highest production of specific immunoglobulin G (IgG), IgG2a, and secretory IgA, where IgG2a was imprinted as a Th1 cell bias. Moreover, this recombinant live vaccine induced significantly high levels of specific cytotoxic T-lymphocyte activities and increased gamma interferon-producing T cells compared with the parental strain. Our work provides insights into how the type IVB pilus operon promoter controlling SARS-CoV N gene expression in Salmonella might be attractive for a live-vector vaccine against SRAS-CoV infection, for it could induce mucosal, humoral, and cellular immune responses. Our work also indicates that the type IVB pilus operon promoter controlling foreign gene expression in Salmonella can elicit full immune responses by intranasal vaccination.

Enhanced Immunity toPlasmodium falciparumCircumsporozoite Protein (PfCSP) by UsingSalmonella entericaSerovar Typhi Expressing PfCSP and a PfCSP-Encoding DNA Vaccine in a Heterologous Prime-Boost Strategy

Two Salmonella enterica serovar Typhi strains that express and export a truncated version of Plasmodium falciparum circumsporozoite surface protein (tCSP) fused to Salmonella serovar Typhi cytolysin A (ClyA) were constructed as a first step in the development of a preerythrocytic malaria vaccine. Synthetic codon-optimized genes (t-csp1 and t-csp2), containing immunodominant B- and T-cell epitopes present in native P. falciparum circumsporozoite surface protein (PfCSP), were fused in frame to the carboxyl terminus of the ClyA gene (clyA::t-csp) in genetically stabilized expression plasmids. Expression and export of ClyA-tCSP1 and ClyA-tCSP2 by Salmonella serovar Typhi vaccine strain CVD 908-htrA were demonstrated by immunoblotting of whole-cell lysates and culture supernatants. The immunogenicity of these constructs was evaluated using a "heterologous prime-boost" approach consisting of mucosal priming with Salmonella serovar Typhi expressing ClyA-tCSP1 and ClyA-tCSP2, followed by parenteral boosting with PfCSP DNA vaccines pVR2510 and pVR2571. Mice primed intranasally on days 0 and 28 with CVD 908-htrA(pSEC10tcsp2) and boosted intradermally on day 56 with PfCSP DNA vaccine pVR2571 induced high titers of serum NANP immunoglobulin G (IgG) (predominantly IgG2a); no serological responses to DNA vaccination were observed in the absence of Salmonella serovar Typhi-PfCSP priming. Mice primed with Salmonella serovar Typhi expressing tCSP2 and boosted with PfCSP DNA also developed high frequencies of gamma interferon-secreting cells, which surpassed those produced by PfCSP DNA in the absence of priming. A prime-boost regimen consisting of mucosal delivery of PfCSP exported from a Salmonella-based live-vector vaccine followed by a parenteral PfCSP DNA boosting is a promising strategy for the development of a live-vector-based malaria vaccine.

Listeria as a vaccine vector

The immunostimulatory characteristics and intracellular niche of Listeria monocytogenes make it uniquely suitable for use as a live bacterial vaccine vector. Preclinical results supporting this idea, and current strategies to induce beneficial cell-mediated immunity to both infectious diseases and cancer with this vector, are discussed in this review.

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope

Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the beta domain of the autotransporter MisL. The passenger alpha domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298-306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein-dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic-dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines.

Role ofd-Alanylation ofStreptococcus gordoniiLipoteichoic Acid in Innate and Adaptive Immunity

In recent years, there has been considerable interest in using the oral commensal gram-positive bacterium Streptococcus gordonii as a live vaccine vector. The present study investigated the role of d-alanylation of lipoteichoic acid (LTA) in the interaction of S. gordonii with the host innate and adaptive immune responses. A mutant strain defective in d-alanylation was generated by inactivation of the dltA gene in a recombinant strain of S. gordonii (PM14) expressing a fragment of the S1 subunit of pertussis toxin. The mutant strain was found to be more susceptible to killing by polymyxin B, nisin, magainin II, and human beta defensins than the parent strain. When it was examined for binding to murine bone marrow-derived dendritic cells (DCs), the dltA mutant exhibited 200- to 400-fold less binding than the parent but similar levels of binding were shown for Toll-like receptor 2 (TLR2) knockout DCs and HEp-2 cells. In a mouse oral colonization study, the mutant showed a colonization ability similar to that of the parent and was not able to induce a significant immune response. The mutant induced significantly less interleukin 12p70 (IL-12p70) and IL-10 than the parent from DCs. LTA purified from the bacteria induced tumor necrosis factor-alpha and IL-6 production from wild-type DCs but not from TLR2 knockout DCs, and the mutant LTA induced a significantly smaller amount of these two cytokines. These results show that d-alanylation of LTA in S. gordonii plays a role in the interaction with the host immune system by contributing to the relative resistance to host defense peptides and by modulating cytokine production by DCs.

Signaling pathways involved in foreign antigen expression by a recombinant vaccinia virus (89.38)

Abstract BACKGROUND: Vaccinia virus (VV) was used as a live vaccine to eradicate smallpox and has in recent years received considerable attention for the development of live vaccine vectors due to its broad host range and ability to express a variety of foreign antigens, which can induce humoral and cellular immunity against virus infections. However, little is known about the cellular signaling pathways involved in the foreign antigen expression by a recombinant VV. METHODS: Using a recombinant EGFP-VV, we studied four cellular signaling pathways, PI3K, p38 MAPK, JNK, and ERK, by assessing EGFP production in infected human peripheral blood monocytes and monocyte-derived dendritic cells (MDDCs). RESULTS: EGFP expression was inhibited significantly in both monocytes and MDDCs infected with EGFP-VV by blocking the PI3K, p38 MAKP, and JNK pathways by specific inhibitors LY294002, SB203580, and SP600125, respectively. PI3K was the most critical pathway involved in antigen expression by VV-infected cells, whereby LY294002 (25 uM) exhibited 90.1% decrease of EGFP expression compared to non-inhibitor-treated control (DMSO). Whereas, at the same concentration (25 uM), SB203580 and SP600125 inhibited only 69.3% and 30.4% of EGFP expression, respectively. Inhibition of the ERK pathway by PD098059 did not alter EGFP expression. CONCLUSIONS: VV is able to infect APCs and efficiently express inserted gene(s) through cellular signaling pathways of PI3K, p38 MAPK, and JNK, but not ERK. ACKNOWLEDGMENTS: This work was supported by University of Washington CFAR (Award AI 27757), Hawaii Community Foundation (Award 20061466), UH INBRE, and HACTU grant from NIAID (A134853).

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.

Enhancement of CD8+ T Cell Immunity in the Lung by CpG Oligodeoxynucleotides Increases Protective Efficacy of a Modified Vaccinia Ankara Vaccine against Lethal Poxvirus Infection Even in a CD4-Deficient Host

Immunostimulatory CpG oligodeoxynucleotides (ODN) have proven effective as adjuvants for protein-based vaccines, but their impact on immune responses induced by live viral vectors is not known. We found that addition of CpG ODN to modified vaccinia Ankara (MVA) markedly improved the induction of longer-lasting adaptive protective immunity in BALB/c mice against intranasal pathogenic vaccinia virus (Western Reserve; WR). Protection was mediated primarily by CD8(+) T cells in the lung, as determined by CD8-depletion studies, protection in B cell-deficient mice, and greater protection correlating with CD8(+) IFN-gamma-producing cells in the lung but not with those in the spleen. Intranasal immunization was more effective at inducing CD8(+) T cell immunity in the lung, and protection, than i.m. immunization. Addition of CpG ODN increased the CD8(+) response but not the Ab response. Depletion of CD4 T cells before vaccination with MVA significantly diminished protection against pathogenic WR virus. However, CpG ODN delivered with MVA was able to substitute for CD4 help and protected CD4-depleted mice against WR vaccinia challenge. This study demonstrates for the first time a protective adjuvant effect of CpG ODN for a live viral vector vaccine that may overcome CD4 deficiency in the induction of protective CD8(+) T cell-mediated immunity.

Nonsegmented Negative-Strand Viruses as Vaccine Vectors

The live-virus vector era began in 1983, when Smith, Mackett, and Moss constructed a recombinant vaccinia virus expressing hepatitis B surface antigen and demonstrated the induction of hepatitis B-specific antibodies in rabbits immunized with the recombinant virus (113). Subsequently, live-virus vectors were developed with other DNA viruses, such as adenoviruses and herpesviruses, and with positive-strand RNA viruses, such as alphaviruses and flaviviruses. The vaccine vector potential of the large group of nonsegmented negative-strand RNA viruses (NNSV) remained unexplored largely due to the lack of infectivity of their genomic RNA in cell culture and the absence of a mechanism for recombinational insertion of foreign genes. The NNSV (order Mononegavirales) comprise four families: Rhabdoviridae, represented by vesicular stomatitis virus (VSV) and rabies virus (RV); Paramyxoviridae, including Sendai virus (SeV), human parainfluenza virus types 1, 2, 3, and 4 (HPIV1 to -4), measles and mumps viruses, Newcastle disease virus (NDV), and human respiratory syncytial and metapneumoviruses (HRSV and HMPV); Filoviridae, containing Ebola and Marburg viruses; and Bornaviridae, containing Borna disease virus. In 1994, Schnell, Mebatsion, and Conzelmann developed a reverse genetic system for RV that allowed the recovery of infectious virus entirely from cloned cDNA (102). This was followed quickly by the development of reverse genetic systems for numerous other NNSV (17, 22, 33, 50, 55, 69, 73, 76, 83, 133). This review will focus on the use of NNSV, in particular members of the Rhabdoviridae and Paramyxoviridae, as live vaccine vectors.