A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

Unboxing dendritic cells: Tales of multi-faceted biology and function.

Often referred to as the bridge between innate and adaptive immunity, dendritic cells (DCs) are professional antigen-presenting cells (APCs) that constitute a unique, yet complex cell system. Among other APCs, DCs display the unique property of inducing protective immune responses against invading microbes, or cancer cells, while safeguarding the proper homeostatic equilibrium of the immune system and maintaining self-tolerance. Unsurprisingly, DCs play a role in many diseases such as autoimmunity, allergy, infectious disease and cancer. This makes them attractive but challenging targets for therapeutics. Since their initial discovery, research and understanding of DC biology have flourished. We now recognize the presence of multiple subsets of DCs distributed across tissues. Recent studies of phenotype and gene expression at the single cell level have identified heterogeneity even within the same DC type, supporting the idea that DCs have evolved to greatly expand the flexibility of the immune system to react appropriately to a wide range of threats. This review is meant to serve as a quick and robust guide to understand the basic divisions of DC subsets and their role in the immune system. Between mice and humans, there are some differences in how these subsets are identified and function, and we will point out specific distinctions as necessary. Throughout the text, we are using both fundamental and therapeutic lens to describe overlaps and distinctions and what this could mean for future research and therapies.

Generation of a reporter yellow fever virus for high throughput antiviral assays.

Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV.

Risk of Zika microcephaly correlates with features of maternal antibodies.

Zika virus (ZIKV) infection during pregnancy causes congenital abnormalities, including microcephaly. However, rates vary widely, and the contributing risk factors remain unclear. We examined the serum antibody response to ZIKV and other flaviviruses in Brazilian women giving birth during the 2015-2016 outbreak. Infected pregnancies with intermediate or higher ZIKV antibody enhancement titers were at increased risk to give birth to microcephalic infants compared with those with lower titers (P < 0.0001). Similarly, analysis of ZIKV-infected pregnant macaques revealed that fetal brain damage was more frequent in mothers with higher enhancement titers. Thus, features of the maternal antibodies are associated with and may contribute to the genesis of ZIKV-associated microcephaly.

Human Genetic Determinants of Viral Diseases.

Much progress has been made in the identification of specific human gene variants that contribute to enhanced susceptibility or resistance to viral diseases. Herein we review multiple discoveries made with genome-wide or candidate gene approaches that have revealed significant insights into virus-host interactions. Genetic factors that have been identified include genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins. We discuss a range of pathogenic viruses, including influenza virus, respiratory syncytial virus, human immunodeficiency virus, human T cell leukemia virus, human papilloma virus, hepatitis B and C viruses, herpes simplex virus, norovirus, rotavirus, parvovirus, and Epstein-Barr virus. Understanding the genetic underpinnings that affect infectious disease outcomes should allow tailored treatment and prevention approaches in the future.

Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex.

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV.IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico.

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.

TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP).

The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.

Interferon regulatory factor 2 protects mice from lethal viral neuroinvasion.

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study, we demonstrate that IFN regulatory factor 2 (IRF2), an ISG and a negative regulator of IFN signaling, influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2-/- mice after peripheral inoculation. Irf2-/- mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2-/- mouse brains despite the presence of peripheral neutralizing antibodies, suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient µMT mice are significantly more susceptible to viral infection, yet WT B cells and serum are unable to rescue the Irf2-/- mice. Collectively, our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections.

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication.

UNLABELLED: DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE: Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.

Identification and quantitation of MHC class II-bound peptides from mouse spleen dendritic cells by immunoprecipitation and mass spectrometry analysis.

Advances in immunology and immune therapies require knowledge of antigenic peptide sequences that are presented on MHC class II and class I molecules of antigen presenting cells. The most specialized antigen presenting cells are dendritic cells (DCs). In the past, the small number of DCs that could be isolated from mouse spleen prevented direct analysis of the MHC II peptide repertoire presented by DCs. Here we describe a protocol that integrates immunological methods (in vivo enrichment of mouse spleen DCs by Flt3L treatment and immunoprecipitation of MHC II-peptide complexes), mass spectrometry analysis and peptide synthesis (LC-MS/MS and quantitation analysis for non tryptic peptides) to identify and quantitate the endogenous peptides that are bound to MHC II molecules on DCs. The described method produces quantitative data that are reproducible and reliable enough to cover a wide range of peptide copy numbers. We propose the application of this method in future studies to quantitatively investigate the MHC II repertoire on DCs presented during viral infections or different immunizations in vaccine development research.

Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.

A new synthetic TLR4 agonist, GLA, allows dendritic cells targeted with antigen to elicit Th1 T-cell immunity in vivo.

Protein-based vaccines offer safety and cost advantages but require adjuvants to induce immunity. Here we examined the adjuvant capacity of glucopyranosyl lipid A (GLA), a new synthetic non-toxic analogue of lipopolysaccharide. In mice, in comparison with non-formulated LPS and monophosphoryl lipid A, formulated GLA induced higher antibody titers and generated Type 1 T-cell responses to HIV gag-p24 protein in spleen and lymph nodes, which was dependent on TLR4 expression. Immunization was greatly improved by targeting HIV gag p24 to DCs with an antibody to DEC-205, a DC receptor for antigen uptake and processing. Subcutaneous immunization induced antigen-specific T-cell responses in the intestinal lamina propria. Immunity did not develop in mice transiently depleted of DCs. To understand how GLA works, we studied DCs directly from vaccinated mice. Within 4 h, GLA caused DCs to upregulate CD86 and CD40 and produce cytokines including IL-12p70 in vivo. Importantly, DCs removed from mice 4 h after vaccination became immunogenic, capable of inducing T-cell immunity upon injection into naïve mice. These data indicate that a synthetic and clinically feasible TLR4 agonist rapidly stimulates full maturation of DCs in vivo, allowing for adaptive immunity to develop many weeks to months later.

Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells.

Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/µL to 12 pmol/µL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.

Improved cellular and humoral immune responses in vivo following targeting of HIV Gag to dendritic cells within human anti-human DEC205 monoclonal antibody.

Protein vaccines for T-cell immunity are not being prioritized because of poor immunogenicity. To overcome this hurdle, proteins are being targeted to maturing dendritic cells (DCs) within monoclonal antibodies (mAbs) to DC receptors. To extend the concept to humans, we immunized human immunoglobulin-expressing mice with human DEC205 (hDEC205) extracellular domain. 3D6 and 3G9 mAbs were selected for high-affinity binding to hDEC205. In addition, CD11c promoter hDEC205 transgenic mice were generated, and 3G9 was selectively targeted to DCs in these animals. When mAb heavy chain was engineered to express HIV Gag p24, the fusion mAb induced interferon-γ- and interleukin-2-producing CD4(+) T cells in hDEC205 transgenic mice, if polynocinic polycytidylic acid was coadministered as an adjuvant. The T-cell response was broad, recognizing at least 3 Gag peptides, and high titers of anti-human immunoglobulin G antibody were made. Anti-hDEC205 also improved the cross-presentation of Gag to primed CD8(+) T cells from HIV-infected individuals. In all tests, 3D6 and 3G9 targeting greatly enhanced immunization relative to nonbinding control mAb. These results provide preclinical evidence that in vivo hDEC205 targeting increases the efficiency with which proteins elicit specific immunity, setting the stage for proof-of-concept studies of these new protein vaccines in human subjects.

HIV gag protein is efficiently cross-presented when targeted with an antibody towards the DEC-205 receptor in Flt3 ligand-mobilized murine DC.

DC present exogenous proteins to MHC class I-restricted CD8+ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8+ T-cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8+ or DEC-205+ DC are specialized for cross-presentation, and this subset can be increased 10-fold in numbers following Fms-like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross-presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti-CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11chigh DC, particularly the DEC-205+ subset. DC cross-present HIV gag to primed CD8+ T cells, but when the antigen is delivered within an antibody to DEC-205 receptor, cross-presentation becomes 100-fold more efficient than non-targeted antigen. This finding requires gag to be engineered into anti-DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross-presentation, since their use overcomes the obstacle posed by the low number of cross-presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC-targeted vaccines.

Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves.

Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter-enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP(+) cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c(+) cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8(+) TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.

Immunotherapy with internally inactivated virus loaded dendritic cells boosts cellular immunity but does not affect feline immunodeficiency virus infection course.

Immunotherapy of feline immunodeficiency virus (FIV)-infected cats with monocyte-derived dendritic cells (MDCs) loaded with aldrithiol-2 (AT2)-inactivated homologous FIV was performed. Although FIV-specific lymphoproliferative responses were markedly increased, viral loads and CD4+ T cell depletion were unaffected, thus indicating that boosting antiviral cell-mediated immunity may not suffice to modify infection course appreciably.

Evaluation of feline monocyte-derived dendritic cells loaded with internally inactivated virus as a vaccine against feline immunodeficiency virus.

Dendritic cells are the only antigen-presenting cells that can present exogenous antigens to both helper and cytolytic T cells and prime Th1-type or Th2-type cellular immune responses. Given their unique immune functions, dendritic cells are considered attractive "live adjuvants" for vaccination and immunotherapy against cancer and infectious diseases. The present study was carried out to assess whether the reinjection of autologous monocyte-derived dendritic cells loaded with an aldithriol-2-inactivated primary isolate of feline immune deficiency virus (FIV) was able to elicit protective immune responses against the homologous virus in naive cats. Vaccine efficacy was assessed by monitoring immune responses and, finally, by challenge with the homologous virus of vaccinated, mock-vaccinated, and healthy cats. The outcome of challenge was followed by measuring cellular and antibody responses and viral and proviral loads and quantitating FIV by isolation and a count of CD4(+)/CD8(+) T cells in blood. Vaccinated animals exhibited clearly evident FIV-specific peripheral blood mononuclear cell proliferation and antibody titers in response to immunization; however, they became infected with the challenge virus at rates comparable to those of control animals.

DEC-205 receptor on dendritic cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes.

Optimal HIV vaccines should elicit CD8+ T cells specific for HIV proteins presented on MHC class I products, because these T cells contribute to host resistance to viruses. We had previously found that the targeting of antigen to dendritic cells (DCs) in mice efficiently induces CD8+ T cell responses. To extend this finding to humans, we introduced the HIV p24 gag protein into a mAb that targets DEC-205/CD205, an endocytic receptor of DCs. We then assessed cross-presentation, which is the processing of nonreplicating internalized antigen onto MHC class I for recognition by CD8+ T cells. Low doses of alphaDEC-gag, but not control Ig-gag, stimulated proliferation and IFN-gamma production by CD8+ T cells isolated from the blood of HIV-infected donors. alphaCD205 fusion mAb was more effective for cross-presentation than alphaCD209/DC-SIGN, another abundant DC uptake receptor. Presentation was diverse, because we identified eight different gag peptides that were recognized via DEC-205 in 11 individuals studied consecutively. Our results, based on humans with highly polymorphic MHC products, reveal that DCs and DEC-205 can cross-present several different peptides from a single protein. Because of the consistency in eliciting CD8+ T cell responses, these data support the testing of alphaDEC-205 fusion mAb as a protein-based vaccine.