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-gamma 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.     

Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes

Vaccine adjuvants such as alum and the oil-in-water emulsion MF59 are used to enhance immune responses towards pure soluble antigens, but their mechanism of action is still largely unclear. Since most adjuvanted vaccines are administered intramuscularly, we studied immune responses in the mouse muscle and found that both adjuvants were potent inducers of chemokine production and promoted rapid recruitment of CD11b⁺ cells. The earliest and most abundantly recruited cell type are neutrophils, followed by monocytes, eosinophils and later dendritic cells (DCs) and macrophages. Using fluorescent forms of MF59 and ovalbumin (OVA) antigen, we show that all recruited cell types take up both adjuvant and antigen to transport them to the draining lymph nodes (LNs). There, we found antigen-positive neutrophils and monocytes within hours of injection, later followed by B cells and DCs. Compared to alum, MF59-injection lead to a more prominent neutrophil recruitment and a more efficient antigen re-localization from the injection site to the LN. As antigen-transporting neutrophils were observed in draining LNs, we asked whether these cells play an essential role in MF59-mediated adjuvanticity. However, antibody-mediated neutrophil ablation left MF59-adjuvanticity unaltered. Further studies will reveal whether other single cell types are crucial or whether the different recruited cell populations are redundant with overlapping functions.     

Alphavirus replicon-based enhancement of mucosal and systemic immunity is linked to the innate response generated by primary immunization

Venezuelan equine encephalitis virus replicon particles (VRP) function as an effective systemic, cellular and mucosal adjuvant when codelivered with antigen, and show promise for use as a component in new and existing human vaccine formulations. We show here that VRP are effective at low dose and by intramuscular delivery, two useful features for implementation of VRP as a vaccine adjuvant. In mice receiving a prime and boost with antigen, we found that VRP are required in prime only to produce a full adjuvant effect. This outcome indicates that the events triggered during prime with VRP are sufficient to establish the nature and magnitude of the immune response to a second exposure to antigen. Events induced by VRP in the draining lymph node after prime include robust secretion of many inflammatory cytokines, upregulation of CD69 on leukocytes, and increased cellularity, with a disproportionate increase of a cell population expressing CD11c, CD11b, and F4/80. We show that antigen delivered 24 h after administration of VRP does not benefit from an adjuvant effect, indicating that the events which are critical to VRP-mediated adjuvant activity occur within the first 24 h. Further studies of the events induced by VRP will help elucidate the mechanism of VRP adjuvant activity and will advance the safe implementation of this adjuvant in human vaccines.     

Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract

The Toll-like receptor 5 (TLR5) agonist flagellin is an effective adjuvant for vaccination. Recently, we demonstrated that the adaptive responses stimulated by intranasal administration of flagellin and antigen were linked to TLR5 signaling in the lung epithelium. The present study sought to identify the antigen presenting cells involved in this adjuvant activity. We first found that the lung dendritic cells captured antigen very efficiently in a process independent of TLR5. However, TLR5-mediated signaling specifically enhanced the maturation of lung dendritic cells. Afterward, the number of antigen-bound and activated conventional dendritic cells (both CD11b⁺ and CD103⁺) increased in the mediastinal lymph nodes in contrast to monocyte-derived dendritic cells. These data suggested that flagellin-activated lung conventional dendritic cells migrate to the draining lymph nodes. The lymph node dendritic cells, in particular CD11b⁺ cells, were essential for induction of CD4 T-cell response. Lastly, neutrophils and monocytes were recruited into the lungs by flagellin administration but did not contribute to the adjuvant activity. The functional activation of conventional dendritic cells was independent of direct TLR5 signaling, thereby supporting the contribution of maturation signals produced by flagellin-stimulated airway epithelium. In conclusion, our results demonstrated that indirect TLR5-dependent stimulation of airway conventional dendritic cells is essential to flagellin's mucosal adjuvant activity.     

Vaccination with liposomal poly(I:C) induces discordant maturation of migratory dendritic cell subsets and anti-viral gene signatures in afferent lymph cells

Vaccine formulations administered in the periphery must activate naive immune cells within the lymph node. In this study, we have directly cannulated the ovine lymphatic vessels to investigate the cellular and molecular mechanisms that transfer information from the periphery into the local draining lymph node via the afferent lymph. Inclusion of poly(I:C) into a liposomal vaccine formulation enhances the neutrophil-associated inflammatory immune response in afferent lymph and increases antigen uptake by migratory dendritic cells (DCs). Interestingly, antigen positive migratory DCs undergo discordant maturation, with peak expression of CD86 at 4 h and CD80 at 48−72 h post vaccination. Afferent lymph monocytes up-regulate expression of genes related to inflammatory and anti-viral immune phenotypes following vaccination however show no differentiation into APCs prior to their migration to the local lymph node as measured by surface MHC II expression. Finally, this study reveals the addition of poly(I:C) increases systemic antigen-specific humoral immunity. These findings provide a detailed understanding of the real time in vivo immune response induced by liposomes incorporating the innate immune agonist poly(I:C) utilising a vaccination setting comparable to that administered in humans.     

Co-localization of a CD1d-binding glycolipid with an adenovirus-based malaria vaccine for a potent adjuvant effect

A CD1d-binding, invariant (i) natural killer T (NKT)-cell stimulatory glycolipid, α-Galactosylceramide (αGalCer), has been shown to act as an adjuvant. We previously identified a fluorinated phenyl ring-modified αGalCer analog, 7DW8-5, displaying a higher binding affinity for CD1d molecule and more potent adjuvant activity than αGalCer. In the present study, 7DW8-5 co-administered intramuscularly (i.m.) with a recombinant adenovirus expressing a Plasmodium yoelii circumsporozoite protein (PyCSP), AdPyCS, has led to a co-localization of 7DW8-5 and a PyCSP in draining lymph nodes (dLNs), particularly in dendritic cells (DCs). This occurrence initiates a cascade of events, such as the recruitment of DCs to dLNs and their activation and maturation, and the enhancement of the ability of DCs to prime CD8+ T cells induced by AdPyCS and ultimately leading to a potent adjuvant effect and protection against malaria.     

The adjuvant PCEP induces recruitment of myeloid and lymphoid cells at the injection site and draining lymph node

Poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) has shown great potential as a vaccine adjuvant, but the mechanisms that mediate its adjuvant activity have not been investigated. Previously, we had reported the potential of PCEP to induce cytokines and chemokines at the site of injection. Hence, we hypothesized that PCEP creates strong immuno-competent environment leading to recruitment of immune cells at the injection site. Intramuscular injection of mice with PCEP induced significant recruitment of neutrophils, macrophages, monocytes, dendritic cells (DCs), and lymphocytes at the site of injection as well as in the draining lymph nodes. Flow cytometric analysis showed that the majority of the recruited immune cells took up and/or were associated with PCEP at the injection site, with lymphocytes taking up PCEP in lesser quantity. Further, confocal analysis revealed intracytoplasmic lysosomal localization of PCEP in recruited immune cells. These observations suggest that recruitment of distinct immune cells to the site of injection site may be an important mechanism by which PCEP potentiates immune responses to antigens.     

TLR8 agonists stimulate newly recruited monocyte-derived cells into potent APCs that enhance HBsAg immunogenicity

We previously reported that synthetic or natural Toll-like receptor (TLR) 7/8 agonists present within dead cells enhanced cell-associated antigen presentation both in vitro and in vivo. Here, we investigated the immunopotency of different chemically synthesized TLR7/8 agonists, Resiquimod, Gardiquimod, CL075, and CL097, on HBsAg immunogenicity. These agonists stimulated inflammatory monocyte-derived cells to become potent antigen-presenting dendritic cells (DCs), which augmented HBsAg specific T cell proliferation after they were conditioned with HBsAg. The TLR8 agonist CL075 and the TLR7/8 dual agonist CL097 showed more potent effects than the TLR7 agonist. Compared with alum adjuvant, when HBsAg mixed with CL075 was injected intramuscularly into mice, more monocyte-derived DCs carried antigens into draining lymph nodes and spleens. Specific Abs, particularly IgG2a, were significantly increased, and more IL-5 and IFN-γ were produced by splenocytes and intrahepatic immunocytes in mice that received HBsAg mixed with CL075 and CL097. These results suggest that TLR8 agonists are good candidates to enhance recombinant HBsAg immunogenicity to induce specific humoral and cellular immune responses.     

Non-standard viral genome-derived RNA activates TLR3 and type I IFN signaling to induce cDC1-dependent CD8+ T-cell responses during vaccination in mice

There is a critical need to develop vaccine adjuvants that induce robust immune responses able to protect against intracellular pathogens, including viruses. Previously, we described defective viral genome-derived oligonucleotides (DDOs) as novel adjuvants that strongly induce type 1 immune responses, including protective Th1 CD4+ T-cells and effector CD8+ T-cells in mice. Here, we unravel the early innate response required for this type 1 immunity induction. Upon DDO subcutaneous injection, type 1 conventional dendritic cells (cDC1s) accumulate rapidly in the draining lymph node in a Toll-like receptor 3 (TLR3)- and type I interferon (IFN)-dependent manner. cDC1 accumulation in the lymph node is required for antigen-specific CD8+ T-cell responses. Notably, in contrast to poly I:C, DDO administration resulted in type I IFN expression at the injection site, but not in the draining lymph node. Additionally, DDOs induced an inflammatory cytokine profile distinct from that induced by poly I:C. Therefore, DDOs represent a powerful new adjuvant to be used during vaccination against intracellular pathogens.     

Comparison of alpha-Type-1 polarizing and standard dendritic cell cytokine cocktail for maturation of therapeutic monocyte-derived dendritic cell preparations from cancer patients

The current "gold standard" for generation of dendritic cell (DC) used in DC-based cancer vaccine studies is maturation of monocyte-derived DCs with tumor necrosis factor-alpha (TNF-alpha)/IL-1beta/IL-6 and prostaglandin E(2) (PGE(2)). Recently, a protocol for producing so-called alpha-Type-1 polarized dendritic cells (alphaDC1) in serum-free medium was published based on maturation of monocyte-derived DCs with TNF-alpha/IL-1-beta/polyinosinic:polycytidylic acid (poly-I:C)/interferon (IFN)-alpha and IFN-gamma. This DC maturation cocktail was described to fulfill the criteria for optimal DC generation and to be superior to the standard DC (sDC) cocktail as it induced fully mature DCs with potent IL-12p70 secretion together with CCR7 expression which is necessary for priming of a TH1 response and for migration to the draining lymph node, respectively. In this study, we tested the adaptation of alphaDC1 maturation cocktail to a protocol for clinical grade DC generation from cancer patients performed in X-VIVO 15 medium. We showed that alphaDC1 in this protocol induce lower up-regulation of CD83 and several other maturation markers, co-stimulatory molecules and CCR7 together with higher up-regulation of inhibitory molecules such as PD-L1, ILT2, ILT3 as compared to sDC. Although alphaDC1 matured DCs secreted more IL-12p70 and IL-23 these DCs had lower or similar stimulatory capacity compared to sDCs when used as stimulating cells in mixed lymphocyte reaction (MLR) or for induction of autologous influenza antigen specific T lymphocytes. Thus, our observations underline that alphaDC1 maturation cannot be directly adapted to alternative protocols for DC generation. Also, this study indicates the necessity for further investigation of correlation between in vitro DC parameters and their in vivo efficacy in clinical vaccination trials.     

Regional,but not systemic recruitment/expansion of dendritic cells by a pluronic-formulated Flt3-ligand plasmid with vaccine adjuvant activity

Regional recruitment of dendritic cells (DCs) by the local administration of granulocyte macrophage-colony stimulating factor (GM-CSF) or Flt3-ligand (Flt3L) has vaccine adjuvant activity. However, Flt3L, with its DC growth factor activity, has not been extensively studied as a vaccine adjuvant, particularly as a plasmid vector. We report that the intramuscular (IM) injection of a Flt3L plasmid (pNGVL-hFlex), when formulated in a pluronic carrier (SP1017, Supratek Pharma, Inc., Laval, Que., Canada), recruits DC to the injection site and regional lymph nodes (LNs) and augments immune responses to a p17 HIV plasmid vaccine to a greater extent than the injection of a naked DNA vaccine alone. Following IM administration of pNGVL-hFlex, Flt3L mRNA, Flt3L protein and infiltrating DC accumulate at the injection site. The number of DC in the draining LNs are also significantly increased with the greatest increase observed following injection of 2.5 microg of pNGVL-hFlex formulated in 0.01% SP1017. Flow cytometric studies demonstrate that the LN-infiltrating DC is mainly of the CD11c(+)CD11b(-) phenotype (IL-12 producing). Further, the co-injection of pNGVL3-hFlex and p17 HIV plasmids, formulated in SP1017, significantly increases the immune responses to the plasmid vaccine (pVAX-gag). The co-injection of pVAX-gag and pNGVL3-hFlex, formulated in SP1017, significantly increase delayed-type hypersensitivity responses and the numbers of antigen (Ag)-specific interferon-gamma secreting T cells in the spleen (Enzyme Linked Immune Spot (ELISpot) assay), compared to mice immunized with pVAX-gag formulated in SP1017 alone. We conclude that the IM injection of pNGVL-hFlex with SP1017 can increase the number of DC in draining LN and at the site of injection, thereby providing adjuvant activity for a plasmid vaccine resulting in a significantly increased, Ag-specific T cell response.     

Induction of local innate immune responses and modulation of antigen uptake as mechanisms underlying the mucosal adjuvant properties of immune stimulating complexes (ISCOMS)

Lipophilic immune stimulating complexes (ISCOMS) containing the Quil A adjuvant can induce local and systemic immune responses against orally delivered protein antigens. Here, we have examined the possibility that ISCOMS act by modulating local innate immune responses and antigen uptake in the intestine. Feeding ISCOMS to mice induced recruitment of dendritic cells (DCs), activated macrophages (mphi) and lymphocytes into the mesenteric lymph node (MLN), as well as recruitment of macrophages and B cells into the PP. Feeding ISCOMS also enhanced the absorption into circulation of a test feed of ovalbumin (OVA), with accelerated kinetics and increased peak levels. Serum taken 1h after feeding a tolerogenic dose of OVA to mice given ISCOMS orally did not induce the tolerance of systemic delayed type hypersensitivity (DTH) and antibody responses found in mice receiving serum from donors fed OVA alone. Thus, ISCOMS may increase the immunogenicity of intestinal antigen via a combination of effects on antigen uptake and on local accessory cells.     

Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based Outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease

Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less immunogenic. The VRP method of delivery induced a Th1 type immune response unlike the recombinant OspA protein in Freund's adjuvant, which induced a mixed (Th1 and Th2) immune response. The VRP tPA-OspA construct induced an immune response that reduced the bacterial load in feeding Ixodes scapularis and blocked transmission to the host. These results indicate that VRPs are capable of providing protection against tick-borne B. burgdorferi, and potentially can be used for developing improved vaccines against Lyme disease.     

Characterization of the innate stimulatory capacity of plant-derived virus-like particles bearing influenza hemagglutinin

Cell-mediated immunity is an important component of immediate and long-term anti-viral protection. Dendritic cells (DCs) are essential for the induction of cell-mediated immunity by instructing the activation and differentiation of antigen-specific T cell responses. Activated DCs that express co-stimulatory molecules and pro-inflammatory cytokines are necessary to promote the development of type 1 immune responses required for viral control. Here we report that plant-derived virus-like particles (VLPs) bearing influenza hemagglutinins (HA) directly stimulate mouse and human DCs. DCs exposed to H1- and, to a lesser extent, H5-VLPs in vitro rapidly express co-stimulatory molecules and produce pro-inflammatory cytokines including IL-12, IL-6 and TNFα. Furthermore, these VLPs support the activation and differentiation of antigen-specific T cell responses. Mechanistically, H1-VLPs stimulate the activation of kinases typically activated downstream of pattern recognition receptors including AKT, p38, and p42/44 ERK. In vivo, immunization with plant-derived VLPs induce the accumulation of both cDC1s and cDC2 in the draining lymph node and a corresponding increase in T and B cells. VLPs devoid of HA protein activate DCs, suggesting they are intrinsically immunostimulatory. Together, the results demonstrate that these candidate plant-derived VLP vaccines have an inherent and direct stimulatory effect on DCs and can enhance the ability of DCs to promote Type 1 immune responses.     

Adjuvant-active aqueous extracts from Artemisia rupestris L. improve immune responses through TLR4 signaling pathway

Activating innate immunity by an adjuvant is required in vaccine development. The study aims to investigate adjuvant effects of aqueous extracts of Artemisia rupestris L. (AEAR) in vivo and in vitro. ICR mice were subcutaneously administered with antigen and AEAR at various doses to evaluate their immune responses of antibodies, dendritic cells (DCs), regulatory T cells (Treg), splenic lymphocyte, and cytokine. The evaluation results showed that AEAR could largely increase titers of antigen-specific antibodies (IgG, IgG₁, and IgG_2a) and T cell proliferation. AEAR also increased expression of IFN-γ in CD8⁺T cells as well as IL-4 and INF-γ expression in CD4⁺T cells. Expression levels of MHC-II, CD40, CD80, and CD86 on DCs were significantly elevated, whereas the Treg frequency was significantly decreased. AEAR (200 μg) showed remarkable adjuvant activity. Furthermore, AEAR enhanced MHC-II, CD40, CD80, and CD86 expression as well as the yields of TNF-α and IL-12 on DCs through toll-like receptor4 (TLR4) in vitro. Those results indicated that AEAR could serve as an efficacious immune stimulator for vaccines because it significantly enhanced specific immune responses by promoting DCs maturation and reduced Treg through TLR4 signaling pathway.     

Analysis of the role of vaccine adjuvants in modulating dendritic cell activation and antigen presentation in vitro

We have studied the effects of adjuvant formulations on the activation and antigen-presenting functions of bone marrow-derived dendritic cells (DCs). While LPS could induce high-level expression of MHC Class II and co-stimulator molecules on DCs, it did not enhance antigen presentation to co-stimulation independent DO11.GFP T hybridoma cells. In contrast, alum, NISV and PLGA formulations failed to activate DCs, but NISV and PLGA could enhance antigen-presentation efficiency by 10-100-fold. Irrespective of the previously described antigen release characteristics of each adjuvant, antigen presentation peaked at 6h and waned thereafter for all formulations. Given the importance of DCs in the activation of nai;ve T cell responses, these studies suggest that as yet undefined pathways of DC activation in vivo may underlie the activity of alum, PLGA and NISV adjuvants. Furthermore, as NISV and PLGA do not appear to act as slow-release systems in DCs, the ability of these particulate systems to induce high levels of antigen presentation by DCs probably has a more significant role in their adjuvant activity.     

Induction of lymphocyte recruitment in the absence of a detectable immune response

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.     

A recombinant Bacille Calmette-Guérin construct expressing the Plasmodium falciparum circumsporozoite protein enhances dendritic cell activation and primes for circumsporozoite-specific memory cells in BALB/c mice

A protective malaria vaccine may induce both high levels of neutralising antibodies and strong T-cell responses. The Plasmodium falciparum circumsporozoite protein (CSp) is a leading pre-erythrocytic vaccine candidate. CSp is a week immunogen per se, but Mycobacterium bovis Bacille Calmette-Guérin (BCG) has excellent adjuvant activity and has been utilized as a vector to deliver heterologous vaccine candidate antigens. It is safe in immunocompetent individuals and inexpensive to produce. We assessed in vitro and in vivo a recombinant BCG-expressing CSp (BCG-CS) as malaria vaccine candidate. Immunisation of BALB/c mice with BCG-CS augmented numbers of dendritic cells (DCs) in draining lymph nodes and in the spleen. The activation markers MHC-class-II, CD40, CD80 and CD86 on DCs were significantly upregulated by BCG-CS as compared to wild-type BCG (wt-BCG). In vitro stimulation of bone marrow-derived DCs and macrophages with BCG-CS induced IL-12 and TNF-α production. BCG-CS induced higher phagocytic activity in macrophages as compared to wt-BCG. Immunogenicity studies show that BCG-CS induced CS-specific antibodies and IFN-γ-producing memory cells. In conclusion, BCG-CS is highly efficient in activating antigen-presenting cells (APCs) for priming of adaptive immunity. Implications for the rational design of novel vaccines against malaria and TB, the two major devastating poverty-related diseases, are discussed.     

Vitamin D3-mediated alterations to myeloid dendritic cell trafficking in vivo expand the scope of their antigen presenting properties

The mucosal immune system provides the host with a first line of adaptive immune defense against invasion by many species of pathogenic microorganisms and their secreted products. Calcitriol, the active form of Vitamin D3 (VD3), promotes the induction of mucosal immunity in mice when added to subcutaneously administered vaccine formulations. Dendritic cells (DCs) activated at vaccination sites where VD3 is present gain the capacity to bypass sequestration in the draining lymph node and traffic to the Peyer's Patches (PP) of immunized animals. By employing protocols that allow the effective tracking of endogenous or adoptively transferred myeloid DCs in vivo, we found that VD3 influences on the trafficking of fully differentiated immature DCs were temporary, and occur without negative effects to antigen processing or peptide presentation to CD4+ T cells. In contrast, DCs differentiated from hematopoietic precursors in the presence of VD3 (conditioned DCs), were markedly compromised in their antigen presenting properties, while manifesting clear alterations to their trafficking properties in vivo. Similar to the recent finding of VD3-mediated enhancement of innate immune protection, our findings suggest that VD3 could also play an important role in controlling the types of immune effector responses elicited subsequent to either infection or vaccination.     

Evaluation of FIV protein-expressing VEE-replicon vaccine vectors in cats

Venezuelan equine encephalitis (VEE) virus-replicon particles (VRP) were used to generate feline immunodeficiency virus (FIV) Gag- and ENV-expressing vaccine vectors. Serum and mucosal FIV-specific antibody was detected in cats immunized subcutaneously, once monthly for 5 months, with FIV-expressing VRP. Expansion of the CD8+ L-selectin negative phenotype and transient CD8+ noncytolytic suppressor activity were seen in cats immunized with FIV-expressing or control VRP. Despite induction of FIV-specific immune responses and nonspecific suppressor responses, all cats became infected following vaginal challenge with high dose, pathogenic cell-associated FIV-NCSU(1) although relative early maintenance of CD4+ cells was seen in FIV-immunized cats.