Readministration of high-dose adeno-associated virus gene therapy vectors enabled by ImmTOR nanoparticles combined with B cell-targeted agents.

Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been demonstrated to mitigate immunogenicity of adeno-associated virus (AAV) gene therapy vectors, enhance levels of transgene expression, and enable redosing of AAV at moderate vector doses of 2 to 5E12 vg/kg. However, recent clinical trials have often pushed AAV vector doses 10-fold to 50-fold higher, with serious adverse events observed at the upper range. Here, we assessed combination therapy of ImmTOR with B cell-targeting drugs for the ability to increase the efficiency of redosing at high vector doses. The combination of ImmTOR with a monoclonal antibody against B cell activation factor (aBAFF) exhibited strong synergy leading to more than a 5-fold to 10-fold reduction of splenic mature B cells and plasmablasts while increasing the fraction of pre-/pro-B cells. In addition, this combination dramatically reduced anti-AAV IgM and IgG antibodies, thus enabling four successive AAV administrations at doses up to 5E12 vg/kg and at least two AAV doses at 5E13 vg/kg, with the transgene expression level in the latter case being equal to that observed in control animals receiving a single vector dose of 1E14 vg/kg. Similar synergistic effects were seen with a combination of ImmTOR and a Bruton's tyrosine kinase inhibitor, ibrutinib. These results suggest that ImmTOR could be combined with B cell-targeting agents to enable repeated vector administrations as a potential strategy to avoid toxicities associated with vector doses above 1E14 vg/kg.

Rapamycin nanoparticles increase the therapeutic window of engineered interleukin-2 and drive expansion of antigen-specific regulatory T cells for protection against autoimmune disease.

Interleukin-2 (IL-2) therapies targeting the high affinity IL-2 receptor expressed on regulatory T cells (Tregs) have shown promising therapeutic benefit in autoimmune diseases through nonselective expansion of pre-existing Treg populations, but are potentially limited by the inability to induce antigen-specific Tregs, as well as by dose-limiting activation of effector immune cells in settings of inflammation. We recently developed biodegradable nanoparticles encapsulating rapamycin, called ImmTOR, which induce selective immune tolerance to co-administered antigens but do not increase total Treg numbers. Here we demonstrate that the combination of ImmTOR and an engineered Treg-selective IL-2 variant (termed IL-2 mutein) increases the number and durability of total Tregs, as well as inducing a profound synergistic increase in antigen-specific Tregs when combined with a target antigen. We demonstrate that the combination of ImmTOR and an IL-2 mutein leads to durable inhibition of antibody responses to co-administered AAV gene therapy capsid, even at sub-optimal doses of ImmTOR, and provides protection in autoimmune models of type 1 diabetes and primary biliary cholangitis. Importantly, ImmTOR also increases the therapeutic window of engineered IL-2 molecules by mitigating effector immune cell expansion and preventing exacerbation of disease in a model of graft-versus-host-disease. At the same time, IL-2 mutein shows potential for dose-sparing of ImmTOR. Overall, these results establish that the combination of ImmTOR and an IL-2 mutein show synergistic benefit on both safety and efficacy to provide durable antigen-specific immune tolerance to mitigate drug immunogenicity and to treat autoimmune diseases.

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

Enhancement of the Tolerogenic Phenotype in the Liver by ImmTOR Nanoparticles.

ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4+ and CD8+ T cells, and the emergence of a large population of CD4-CD8- (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment.

Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles.

Systemic AAV (adeno-associated virus) gene therapy is a promising approach for the treatment of inborn errors of metabolism, but questions remain regarding its potency and durability. Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been shown to block the formation of neutralizing anti-capsid antibodies, thereby enabling vector re-administration. Here, we further demonstrate that ImmTOR admixed with AAV vectors also enhances hepatic transgene expression at the initial dose of AAV vector, independent of its effects on adaptive immunity. ImmTOR enhances AAV trafficking to the liver, resulting in increased hepatic vector copy numbers and transgene mRNA expression. Enhanced transgene expression occurs through a mechanism independent of the AAV receptor and cannot be replicated in vivo with free rapamycin or empty nanoparticles. The multipronged mechanism of ImmTOR action makes it an attractive candidate to enable more efficient transgene expression at first dose while simultaneously inhibiting adaptive responses against AAV to enable repeat dosing.

Long-term correction of ornithine transcarbamylase deficiency in Spf-Ash mice with a translationally optimized AAV vector.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTCSpf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic.

Novel Prefrontal Synthesis Intervention Improves Language in Children with Autism.

Prefrontal synthesis (PFS) is defined as the ability to juxtapose mental visuospatial objects at will. Paralysis of PFS may be responsible for the lack of comprehension of spatial prepositions, semantically-reversible sentences, and recursive sentences observed in 30 to 40% of individuals with autism spectrum disorder (ASD). In this report we present data from a three-year-long clinical trial of 6454 ASD children age 2 to 12 years, which were administered a PFS-targeting intervention. Tablet-based verbal and nonverbal exercises emphasizing mental-juxtaposition-of-objects were organized into an application called Mental Imagery Therapy for Autism (MITA). The test group included participants who completed more than one thousand exercises and made no more than one error per exercise. The control group was selected from the rest of participants by a matching procedure. Each test group participant was matched to the control group participant by age, gender, expressive language, receptive language, sociability, cognitive awareness, and health score at first evaluation using propensity score analysis. The test group showed a 2.2-fold improvement in receptive language score vs. control group (p < 0.0001) and a 1.4-fold improvement in expressive language (p = 0.0144). No statistically significant change was detected in other subscales not targeted by the exercises. These findings show that language acquisition improves after training PFS and that a further investigation of the PFS-targeting intervention in a randomized controlled study is warranted.

Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration.

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration.

Synthetic vaccine particles for durable cytolytic T lymphocyte responses and anti-tumor immunotherapy.

We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1-2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13-14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.

Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release.

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required.

Generation of a universal CD4 memory T cell recall peptide effective in humans, mice and non-human primates.

CD4T cells play a key role in humoral immunity by providing help to B cells, enabling effective antibody class switching and affinity maturation. Some vaccines may generate a poor response due to a lack of effective MHC class II epitopes, resulting in ineffective helper T cell activation and recall and consequently poor humoral immunity. It may be beneficial to provide a CD4T cell helper peptide with a vaccine particularly in the case of a poorly immunogenic antigen. Such a T cell helper peptide must be promiscuous in its ability to bind a broad range of MHC class II alleles due to broad allelic variation in the human population. We designed a chimeric MHC class II peptide (TpD) with epitopes from tetanus toxoid and diphtheria toxoid, separated by an internal cathepsin cleavage site. TpD was capable of inducing a memory recall response in peripheral blood mononuclear cells from 20/20 human donors. T cells responding to TpD showed a central memory phenotype. Immunization of mice with a synthetic nicotine nanoparticle vaccine containing TpD showed that the peptide was required for robust antibody production and resulted in a long term CD4 memory T cell recall response. As a pre-clinical model two non-human primate species, rhesus macaques and cynomolgus monkeys, were immunized with a nicotine nanoparticle vaccine and evaluated for an anti-nicotine antibody response and TpD specific memory T cells. We found that 4/4 rhesus monkeys had both sustained antibody production and TpD memory T cells for the duration of the experiment (119 days). In addition 30/30 cynomolgus monkeys dosed with nicotine vaccine nanoparticles showed dose-dependent antibody generation and T cell recall response compared to saline injected controls. In summary we have developed a potent universal memory T cell helper peptide (TpD) that is active in vitro in human PBMCs and in vivo in mice and non-human primates.

Importance of mRNA secondary structural elements for the expression of influenza virus genes.

Development of novel vaccines and therapeutics often requires efficient expression of recombinant viral proteins. Here we show that mutations in essential functional regions of conserved influenza proteins NP and NS1, lead to reduced expression of these genes in vitro. According to in silico analysis, these mRNA regions possess distinct secondary structures sensitive to mutations. We identified a novel structural feature within a region in NS1 mRNA that encodes amino acids essential for NS1 function. Mutations altering this mRNA element lead to significantly reduced protein expression. Conversely, expression was not affected by mutations resulting in amino acid substitutions, when they were designed to preserve this secondary RNA structural element. Furthermore, altering this structure significantly reduced RNA transcription without affecting mRNA stability. Therefore, distinct internal secondary structures of viral mRNA may be important for viral gene expression. If such elements encode amino acids essential for the protein function, then early selection against mutations in this region will be beneficial for the virus. This might point at yet another mechanism of viral evolution, especially for RNA viruses. Finally, introducing mutations into viral genes while preserving their secondary RNA structure, suggests a new method for the generation of efficiently expressed recombinants of viral proteins.

Effective expression of recombinant cytotoxic protein via its attachment to a polyglutamine domain.

Inadvertent cytotoxicity may hinder the expression of many recombinant proteins that are of industrial or medicinal importance. Here, we show that covalent binding of the influenza A cytotoxic protein M2 to a polyglutamine domain (polyQ-M2; QM2) results in significant delay of its cytotoxic effects when compared to wild-type protein (M2wt). We also show that while expression of recombinant M2wt from A/WSN/1933 strain could not be attained in vaccinia virus (VV), polyQ-M2 was successfully expressed in this system. Moreover, we demonstrate that in cell culture, the polyQ domain is cleaved off following 48 h of expression, thus releasing free and active M2. Similarly, we show the spontaneous cleavage and polyQ release from fusion with another distinct polypeptide, green fluorescent protein (GFP). Expression of M2 from QM2 construct was more prolonged than one based on M2wt-expressing construct, markedly exceeding it at the later time points. Therefore, cell death caused by a toxic polypeptide may be suppressed via genetic fusion with polyQ, resulting in its enhanced expression, followed by slow release of the free polypeptide from the fusion. Collectively, covalent fusion with polyQ or other aggregate-forming domains presents a novel approach for industrial production of cytotoxic proteins and also holds promise for gene therapy applications.

Development of a vaccine against pandemic influenza viruses: current status and perspectives.

The constant threat of a new influenza pandemic, which may be caused by a highly pathogenic avian influenza virus, necessitates the development of a vaccine capable of providing efficient, long-term, and cost-effective protection. Proven avenues for the development of vaccines against seasonal influenza as well as novel approaches have been explored over the past decade. Whereas significant insights are consistently being made, the generation of a highly efficient and cross-protective vaccine against the future pandemic influenza strain remains as the ultimate goal in the field. In this review, we re-examine these efforts and outline the scientific, political, and economic problems that befall this area of biotechnological research.

The proteosomal degradation of fusion proteins cannot be predicted from the proteosome susceptibility of their individual components.

It is assumed that the proteosome-processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome-degradable proteins does not necessarily result in a proteosome-degradable chimera. Conversely, fusion of proteosome-resistant proteins may result in a proteosome-degradable composite. We previously demonstrated that conserved influenza proteins can be unified into a single fusion antigen that is protective, and that vaccination with combinations of proteosome-resistant and proteosome-degradable antigens resulted in an augmented T-cell response. In the present study we constructed proteosome-degradable mutants of conserved influenza proteins NP, M1, NS1, and M2. These were then fused into multipartite proteins in different positions. The stability and degradation profiles of these fusion constructs were demonstrated to depend on the relative position of the individual proteins within the chimeric molecule. Combining unstable sequences of either NP and M1 or NS1 and M2 resulted in either rapidly proteosome degraded or proteosome-resistant bipartite fusion mutants. However, further unification of the proteosome-degradable forms into a single four-partite fusion molecule resulted in relatively stable chimeric proteins. Conversely, the addition of proteosome-resistant wild-type M2 to proteosome-resistant NP-M1-NS1 fusion protein lead to the decreased stability of the resulting four-partite multigene products, which in one case was clearly proteosome dependent. Additionally, a highly destabilized form of M1 failed to destabilize the wild-type NP. Collectively, we did not observe any additive effect leading to proteosomal degradation/nondegradation of a multigene construct.

Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency.

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed.

Toxicity of influenza A virus matrix protein 2 for mammalian cells is associated with its intrinsic proton-channeling activity.

Molecules of influenza matrix protein 2 (M2) are organized in tetramers that constitute a well-conserved virion component and also form proton channels in the plasma membrane of infected cells. In this report we demonstrate that influenza M2 protein is cytopathic in vitro for mammalian cells. An M2 point-mutant (M2pm) protein was constructed that contained amino acid changes designed to block the proton channel via introduction of large hydrophobic residues. This mutant was significantly less toxic upon transient transfection in vitro than the wild-type M2 (M2wt). To assess the possible correlation between M2 cytotoxicity and its proton channel activity, we monitored changes in mitochondria membrane potential induced by M2wt and M2pm. M2wt rapidly decreased mitochondria membrane potential reflecting the transmembrane proton gradient, while M2pm was markedly less efficient. Thus, M2 is cytotoxic for mammalian cells, likely via its proton channel activity and may therefore contribute to influenza pathogenesis through this previously unknown mechanism.

Protection against mouse and avian influenza A strains via vaccination with a combination of conserved proteins NP, M1 and NS1.

BACKGROUND: Experimental data accumulated over more than a decade indicate that cross-strain protection against influenza may be achieved by immunization with conserved influenza proteins. At the same time, the efficacy of immunization schemes designed along these lines and involving internal influenza proteins, mostly NP and M1, has not been sufficient. OBJECTIVE: To test the immunogenicity and protective efficacy of DNA vaccination with a combination of NP, M1 and NS1 genes of influenza virus. METHODS: The immunogenicity and protective efficacy of DNA vaccination with NP, M1 and NS1 was tested in mice and chickens. Mice were challenged with mouse-adapted viral strains H3N2 and H5N2 and chicken challenged with avian H5N3 virus. RESULTS: In these settings, wild-type NS1 did not impede the cellular and humoral response to NP/M1 immunization in vivo. Moreover, addition of NS1-encoding plasmid to the NP/M1 immunization protocol resulted in a significantly increased protective efficacy in vivo. CONCLUSIONS: The addition of NS1 to an influenza immunization regimen based on conserved proteins bears promise. It is feasible that upon further genetic modification of these and additional conserved influenza proteins, providing for their higher safety, expression and immunogenicity, a recombinant vaccine based on several structural and non-structural proteins or their epitopes will offer broad anti-influenza protection in a wide range of species.

CD1d mediates T-cell-dependent resistance to secondary infection with encephalomyocarditis virus (EMCV) in vitro and immune response to EMCV infection in vivo.

The innate and adaptive immune responses have evolved distinct strategies for controlling different viral pathogens. Encephalomyocarditis virus (EMCV) is a picornavirus that can cause paralysis, diabetes, and myocarditis within days of infection. The optimal innate immune response against EMCV in vivo requires CD1d. Interaction of antigen-presenting cell CD1d with distinct natural killer T-cell ("NKT") populations can induce rapid gamma interferon (IFN-gamma) production and NK-cell activation. The T-cell response of CD1d-deficient mice (lacking all NKT cells) against acute EMCV infection was further studied in vitro and in vivo. EMCV persisted at higher levels in CD1d-knockout (KO) splenocyte cultures infected in vitro. Furthermore, optimal resistance to repeat cycles of EMCV infection in vitro was also shown to depend on CD1d. However, this was not reflected in the relative levels of NK-cell activation but rather by the responses of both CD4(+) and CD8(+) T-cell populations. Repeated EMCV infection in vitro induced less IFN-gamma and alpha interferon (IFN-alpha) from CD1d-deficient splenocytes than with the wild type. Furthermore, the level of EMCV replication in wild-type splenocytes was markedly and specifically increased by addition of blocking anti-CD1d antibody. Depletion experiments demonstrated that dendritic cells contributed less than the combination of NK and NKT cells to anti-EMCV responses and that none of these cell types was the main source of IFN-alpha. Finally, EMCV infection in vivo produced higher levels of viremia in CD1d-KO mice than in wild-type animals, coupled with significantly less lymphocyte activation and IFN-alpha production. These results point to the existence of a previously unrecognized mechanism of rapid CD1d-dependent stimulation of the antiviral adaptive cellular immune response.

Role of genes that modulate host immune responses in the immunogenicity and pathogenicity of vaccinia virus.

Poxvirus vaccine vectors, although capable of eliciting potent immune responses, pose serious health risks in immunosuppressed individuals. We therefore constructed five novel recombinant vaccinia virus vectors which contained overlapping deletions of coding regions for the B5R, B8R, B12R, B13R, B14R, B16R, B18R, and B19R immunomodulatory gene products and assessed them for both immunogenicity and pathogenicity. All five of these novel vectors elicited both cellular and humoral immunity to the inserted HIV-BH10 env comparable to that induced by the parental Wyeth strain vaccinia virus. However, deletion of these immunomodulatory genes did not increase the immunogenicity of these vectors compared with the parental vaccinia virus. Furthermore, four of these vectors were slightly less virulent and one was slightly more virulent than the Wyeth strain virus in neonatal mice. Attenuated poxviruses have potential use as safer alternatives to current replication-competent vaccinia virus. Improved vaccinia virus vectors can be generated by deleting additional genes to achieve a more significant viral attenuation.