The vaccinia-based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses.

The ongoing coronavirus disease 2019 (COVID-19) pandemic perpetuated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has highlighted the continued need for broadly protective vaccines that elicit robust and durable protection. Here, the vaccinia virus-based, replication-defective Sementis Copenhagen Vector (SCV) was used to develop a first-generation COVID-19 vaccine encoding the spike glycoprotein (SCV-S). Vaccination of mice rapidly induced polyfunctional CD8 T cells with cytotoxic activity and robust type 1 T helper-biased, spike-specific antibodies, which are significantly increased following a second vaccination, and contained neutralizing activity against the alpha and beta variants of concern. Longitudinal studies indicated that neutralizing antibody activity was maintained up to 9 months after vaccination in both young and middle-aged mice, with durable immune memory evident even in the presence of pre-existing vector immunity. Therefore, SCV-S vaccination has a positive immunogenicity profile, with potential to expand protection generated by current vaccines in a heterologous boost format and presents a solid basis for second-generation SCV-based COVID-19 vaccine candidates incorporating additional SARS-CoV-2 immunogens.

Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation.

CDK8 regulates transcription either by phosphorylation of transcription factors or, as part of a four-subunit kinase module, through a reversible association of the kinase module with the Mediator complex, a highly conserved transcriptional coactivator. Deregulation of CDK8 has been found in various types of human cancer, while the role of CDK8 in supressing anti-cancer response of natural killer cells is being understood. Currently, CDK8-targeting cancer drugs are highly sought-after. Herein we detail the discovery of a series of novel pyridine-derived CDK8 inhibitors. Medicinal chemistry optimisation gave rise to 38 (AU1-100), a potent CDK8 inhibitor with oral bioavailability. The compound inhibited the proliferation of MV4-11 acute myeloid leukaemia cells with the kinase activity of cellular CDK8 dampened. No systemic toxicology was observed in the mice treated with 38. These results warrant further pre-clinical studies of 38 as an anti-cancer agent.

The vaccinia virus based Sementis Copenhagen Vector vaccine against Zika and chikungunya is immunogenic in non-human primates.

The Sementis Copenhagen Vector (SCV) is a new vaccinia virus-derived, multiplication-defective, vaccine technology assessed herein in non-human primates. Indian rhesus macaques (Macaca mulatta) were vaccinated with a multi-pathogen recombinant SCV vaccine encoding the structural polyproteins of both Zika virus (ZIKV) and chikungunya virus (CHIKV). After one vaccination, neutralising antibody responses to ZIKV and four strains of CHIKV, representative of distinct viral genotypes, were generated. A second vaccination resulted in significant boosting of neutralising antibody responses to ZIKV and CHIKV. Following challenge with ZIKV, SCV-ZIKA/CHIK-vaccinated animals showed significant reductions in viremias compared with animals that had received a control SCV vaccine. Two SCV vaccinations also generated neutralising and IgG ELISA antibody responses to vaccinia virus. These results demonstrate effective induction of immunity in non-human primates by a recombinant SCV vaccine and illustrates the utility of SCV as a multi-disease vaccine platform capable of delivering multiple large immunogens.

Aberrant Expression of High Mobility Group Box Protein 1 in the Idiopathic Inflammatory Myopathies.

INTRODUCTION: High Mobility Group Box Protein 1 (HMGB1) is a DNA-binding protein that exerts inflammatory or pro-repair effects upon translocation from the nucleus. We postulate aberrant HMGB1 expression in immune-mediated necrotising myopathy (IMNM). METHODS: Herein, we compare HMGB1 expression (serological and sarcoplasmic) in patients with IMNM with that of other myositis subtypes using immunohistochemistry and ELISA. RESULTS: IMNM (n = 62) and inclusion body myositis (IBM, n = 14) patients had increased sarcoplasmic HMGB1 compared with other myositis patients (n = 46). Sarcoplasmic HMGB1 expression correlated with muscle weakness and histological myonecrosis, inflammation, regeneration and autophagy. Serum HMGB1 levels were elevated in patients with IMNM, dermatomyositis and polymositis, and those myositis patients with extramuscular inflammatory features. DISCUSSION: Aberrant HMGB1 expression occurs in myositis patients and correlates with weakness. A unique expression profile of elevated sarcoplasmic and serum HMGB1 was detected in IMNM.

Thymus-Derived Regulatory T Cells Exhibit Foxp3 Epigenetic Modification and Phenotype Attenuation after Mating in Mice.

Regulatory T cells (Tregs) are essential for maternal tolerance in allogeneic pregnancy. In preeclampsia, Tregs are fewer and display aberrant phenotypes, particularly in the thymic Treg (tTreg) compartment, potentially because of insufficient priming to male partner alloantigens before conception. To investigate how tTregs as well as peripheral Tregs (pTregs) respond to male partner seminal fluid, Foxp3+CD4+ Tregs were examined in the uterus and uterus-draining lymph nodes in virgin estrus mice and 3.5 d postcoitum. Mating elicited 5-fold increases in uterine Tregs accompanied by extensive Treg proliferation in the uterus-draining lymph nodes, comprising 70% neuropilin 1+ tTregs and 30% neuropilin 1- pTregs. Proliferation marker Ki67 and suppressive competence markers Foxp3 and CTLA4 were induced after mating in both subsets, and Ki67, CTLA4, CD25, and GITR were higher in tTregs than in pTregs. Analysis by t-stochastic neighbor embedding confirmed phenotypically distinct tTreg and pTreg clusters, with the proportion of tTregs but not pTregs among CD4+ T cells expanding in response to seminal fluid. Bisulphite sequencing revealed increased demethylation of the Treg-specific demethylation region in the Foxp3 locus in tTregs but not pTregs after mating. These data show that tTregs and pTregs with distinct phenotypes both respond to seminal fluid priming, but the Foxp3 epigenetic signature is uniquely increased in tTregs. We conclude that reproductive tract tTregs as well as pTregs are sensitive to local regulation by seminal fluid, providing a candidate mechanism warranting evaluation for the potential to influence preeclampsia susceptibility in women.

Viral Replicative Capacity, Antigen Availability via Hematogenous Spread, and High TFH:TFR Ratios Drive Induction of Potent Neutralizing Antibody Responses.

Live viral vaccines elicit protective, long-lived humoral immunity, but the underlying mechanisms through which this occurs are not fully elucidated. Generation of affinity matured, long-lived protective antibody responses involve close interactions between T follicular helper (TFH) cells, germinal center (GC) B cells, and T follicular regulatory (TFR) cells. We postulated that escalating concentrations of antigens from replicating viruses or live vaccines, spread through the hematogenous route, are essential for the induction and maintenance of long-lived protective antibody responses. Using replicating and poorly replicating or nonreplicating orthopox and influenza A viruses, we show that the magnitude of TFH cell, GC B cell, and neutralizing antibody responses is directly related to virus replicative capacity. Further, we have identified that both lymphoid and circulating TFH:TFR cell ratios during the peak GC response can be used as an early predictor of protective, long-lived antibody response induction. Finally, administration of poorly or nonreplicating viruses to allow hematogenous spread generates significantly stronger TFH:TFR ratios and robust TFH, GC B cell and neutralizing antibody responses.IMPORTANCE Neutralizing antibody response is the best-known correlate of long-term protective immunity for most of the currently licensed clinically effective viral vaccines. However, the host immune and viral factors that are critical for the induction of robust and durable antiviral humoral immune responses are not well understood. Our study provides insight into the dynamics of key cellular mediators of germinal center reaction during live virus infections and the influence of viral replicative capacity on the magnitude of antiviral antibody response and effector function. The significance of our study lies in two key findings. First, the systemic spread of even poorly replicating or nonreplicating viruses to mimic the spread of antigens from replicating viruses due to escalating antigen concentration is fundamental to the induction of durable antibody responses. Second, the TFH:TFR ratio may be used as an early predictor of protective antiviral humoral immune responses long before memory responses are generated.

Uterine B Cells Exhibit Regulatory Properties During the Peri-Implantation Stage of Murine Pregnancy.

A successful outcome to pregnancy is dependent on the ability of the maternal uterine microenvironment to regulate inflammation processes and establish maternal tolerance. Recently, B cells have been shown to influence pregnancy outcomes as aberrations in their numbers and functions are associated with obstetric complications. In this study, we aimed to comprehensively examine the population frequency and phenotypic profile of B cells over the course of murine pregnancy. Our results demonstrated a significant expansion in B cells within the uterus during the peri-implantation period, accompanied by alterations in B cell phenotype. Functional evaluation of uterine B cells purified from pregnant mice at day 5.5 post-coitus established their regulatory capacity as evidenced by effective suppression of proliferation and activation of syngeneic CD4+ T cells. Flow cytometric analysis revealed that the uterine B cell population has an expanded pool of IL-10-producing B cells bearing upregulated expression of co-stimulatory molecules CD80 and CD86 and activation marker CD27. Our investigations herein demonstrate that during the critical stages surrounding implantation, uterine B cells are amplified and phenotypically modified to act in a regulatory manner that potentially contributes toward the establishment of maternal immunological tolerance in early pregnancy.

Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo.

Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.

A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses.

Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR-/- mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR-/- mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR-/- mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.

Targeting prostate cancer cells with hybrid elastin-like polypeptide/liposome nanoparticles.

Prostate cancer cells frequently overexpress the gastrin-releasing peptide receptor, and various strategies have been applied in preclinical settings to target this receptor for the specific delivery of anticancer compounds. Recently, elastin-like polypeptide (ELP)-based self-assembling micelles with tethered GRP on the surface have been suggested to actively target prostate cancer cells. Poorly soluble chemotherapeutics such as docetaxel (DTX) can be loaded into the hydrophobic cores of ELP micelles, but only limited drug retention times have been achieved. Herein, we report the generation of hybrid ELP/liposome nanoparticles which self-assembled rapidly in response to temperature change, encapsulated DTX at high concentrations with slow release, displayed the GRP ligand on the surface, and specifically bound to GRP receptor expressing PC-3 cells as demonstrated by flow cytometry. This novel type of drug nanocarrier was successfully used to reduce cell viability of prostate cancer cells in vitro through the specific delivery of DTX.

Cytolytic DNA vaccine encoding lytic perforin augments the maturation of- and antigen presentation by- dendritic cells in a time-dependent manner.

The use of cost-effective vaccines capable of inducing robust CD8+ T cell immunity will contribute significantly towards the elimination of persistent viral infections and cancers worldwide. We have previously reported that a cytolytic DNA vaccine encoding an immunogen and a truncated mouse perforin (PRF) protein significantly augments anti-viral T cell (including CD8+ T cell) immunity. Thus, the current study investigated whether this vaccine enhances activation of dendritic cells (DCs) resulting in greater priming of CD8+ T cell immunity. In vitro data showed that transfection of HEK293T cells with the cytolytic DNA resulted in the release of lactate dehydrogenase, indicative of necrotic/lytic cell death. In vitro exposure of this lytic cell debris to purified DCs from naïve C57BL/6 mice resulted in maturation of DCs as determined by up-regulation of CD80/CD86. Using activation/proliferation of adoptively transferred OT-I CD8+ T cells to measure antigen presentation by DCs in vivo, it was determined that cytolytic DNA immunisation resulted in a time-dependent increase in the proliferation of OT-I CD8+ T cells compared to canonical DNA immunisation. Overall, the data suggest that the cytolytic DNA vaccine increases the activity of DCs which has important implications for the design of DNA vaccines to improve their translational prospects.

Production of a Chikungunya Vaccine Using a CHO Cell and Attenuated Viral-Based Platform Technology.

Vaccinia-based systems have been extensively explored for the development of recombinant vaccines. Herein we describe an innovative vaccinia virus (VACV)-derived vaccine platform technology termed Sementis Copenhagen Vector (SCV), which was rendered multiplication-defective by targeted deletion of the essential viral assembly gene D13L. A SCV cell substrate line was developed for SCV vaccine production by engineering CHO cells to express D13 and the VACV host-range factor CP77, because CHO cells are routinely used for manufacture of biologics. To illustrate the utility of the platform technology, a SCV vaccine against chikungunya virus (SCV-CHIK) was developed and shown to be multiplication-defective in a range of human cell lines and in immunocompromised mice. A single vaccination of mice with SCV-CHIK induced antibody responses specific for chikungunya virus (CHIKV) that were similar to those raised following vaccination with a replication-competent VACV-CHIK and able to neutralize CHIKV. Vaccination also provided protection against CHIKV challenge, preventing both viremia and arthritis. Moreover, SCV retained capacity as an effective mouse smallpox vaccine. In summary, SCV represents a new and safe vaccine platform technology that can be manufactured in modified CHO cells, with pre-clinical evaluation illustrating utility for CHIKV vaccine design and construction.

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

Targeting prostate cancer cells with genetically engineered polypeptide-based micelles displaying gastrin-releasing peptide.

In recent years G protein-coupled receptors (GPCRs) have emerged as crucial tumorigenic factors that drive aberrant cancer growth, metastasis and angiogenesis. Consequently, a number of GPCRs are strongly expressed in cancer derived cell lines and tissue samples. Therefore a rational anti-cancer strategy is the design of nano-medicines that specifically target GPCRs to bind and internalise cytotoxic drugs into cancer cells. Herein, we report the genetic engineering of a self-assembling nanoparticle based on elastin-like polypeptide (ELP), which has been fused with gastrin releasing peptide (GRP). These nanoparticles increased intracellular calcium concentrations when added to GRP receptor positive PC-3 prostate cancer cells, demonstrating specific receptor activation. Moreover, GRP-displaying fluorescent labelled nanoparticles showed specific cell-surface interaction with PC-3 prostate cancer cells and increased endocytic uptake. These nanoparticles therefore provide a targeted molecular carrier system for evaluating the delivery of cytotoxic drugs into cancer cells.

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naïve Mice.

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.

Deficiency in Th2 cytokine responses exacerbate orthopoxvirus infection.

Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbpΔ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbpΔ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.

Vaccine-induced protection against orthopoxvirus infection is mediated through the combined functions of CD4 T cell-dependent antibody and CD8 T cell responses.

UNLABELLED: Antibody production by B cells in the absence of CD4 T cell help has been shown to be necessary and sufficient for protection against secondary orthopoxvirus (OPV) infections. This conclusion is based on short-term depletion of leukocyte subsets in vaccinated animals, in addition to passive transfer of immune serum to naive hosts that are subsequently protected from lethal orthopoxvirus infection. Here, we show that CD4 T cell help is necessary for neutralizing antibody production and virus control during a secondary ectromelia virus (ECTV) infection. A crucial role for CD4 T cells was revealed when depletion of this subset was extended beyond the acute phase of infection. Sustained depletion of CD4 T cells over several weeks in vaccinated animals during a secondary infection resulted in gradual diminution of B cell responses, including neutralizing antibody, contemporaneous with a corresponding increase in the viral load. Long-term elimination of CD8 T cells alone delayed virus clearance, but prolonged depletion of both CD4 and CD8 T cells resulted in death associated with uncontrolled virus replication. In the absence of CD4 T cells, perforin- and granzyme A- and B-dependent effector functions of CD8 T cells became critical. Our data therefore show that both CD4 T cell help for antibody production and CD8 T cell effector function are critical for protection against secondary OPV infection. These results are consistent with the notion that the effectiveness of the smallpox vaccine is related to its capacity to induce both B and T cell memory. IMPORTANCE: Smallpox eradication through vaccination is one of the most successful public health endeavors of modern medicine. The use of various orthopoxvirus (OPV) models to elucidate correlates of vaccine-induced protective immunity showed that antibody is critical for protection against secondary infection, whereas the role of T cells is unclear. Short-term leukocyte subset depletion in vaccinated animals or transfer of immune serum to naive, immunocompetent hosts indicates that antibody alone is necessary and sufficient for protection. We show here that long-term depletion of CD4 T cells over several weeks in vaccinated animals during secondary OPV challenge reveals an important role for CD4 T cell-dependent antibody responses in effective virus control. Prolonged elimination of CD8 T cells alone delayed virus clearance, but depletion of both T cell subsets resulted in death associated with uncontrolled virus replication. Thus, vaccinated individuals who subsequently acquire T cell deficiencies may not be protected against secondary OPV infection.

A natural genetic variant of granzyme B confers lethality to a common viral infection.

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining 'Asp-ase' activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmBW mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.

The orchestrated functions of innate leukocytes and T cell subsets contribute to humoral immunity, virus control, and recovery from secondary poxvirus challenge.

A pivotal role for antigen-specific recall responses to secondary virus infection is well established, but the contribution of innate immune cells to this process is unknown. Recovery of mice from a primary orthopoxvirus (ectromelia virus [ECTV]) infection requires the function of natural killer (NK) cells, granulocytes, plasmacytoid dendritic cells (pDC), T cells, and B cells. However, during a secondary challenge, resolution of infection is thought to be dependent on antibody but not T cell function. We investigated the contribution of NK cells, granulocytes, and pDC to virus control during a secondary virus challenge in mice that had been primed with an avirulent, mutant strain of ECTV. Mice depleted of NK cells, granulocytes, or pDC effectively controlled virus, as did mice depleted of both CD4 and CD8 T cell subsets. However, mice concurrently depleted of all three innate cell subsets had elevated virus load, but this was significantly exacerbated in mice also depleted of CD4 and/or CD8 T cells. Increased viral replication in mice lacking innate cells plus CD4 T cells was associated with a significant reduction in neutralizing antibody. Importantly, in addition to T-dependent neutralizing antibody responses, the function of CD8 T cells was also clearly important for virus control. The data indicate that in the absence of innate cell subsets, a critical role for both CD4 and CD8 T cells becomes apparent and, conversely, in the absence of T cell subsets, innate immune cells help contain infection.