Improved Durability to SARS-CoV-2 Vaccine Immunity following Coimmunization with Molecular Adjuvant Adenosine Deaminase-1.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have demonstrated strong immunogenicity and protection against severe disease, concerns about the duration and breadth of these responses remain. In this study, we show that codelivery of plasmid-encoded adenosine deaminase-1 (pADA) with SARS-CoV-2 spike glycoprotein DNA enhances immune memory and durability in vivo. Coimmunized mice displayed increased spike-specific IgG of higher affinity and neutralizing capacity as compared with plasmid-encoded spike-only-immunized animals. Importantly, pADA significantly improved the longevity of these enhanced responses in vivo. This coincided with durable increases in frequencies of plasmablasts, receptor-binding domain-specific memory B cells, and SARS-CoV-2-specific T follicular helper cells. Increased spike-specific T cell polyfunctionality was also observed. Notably, animals coimmunized with pADA had significantly reduced viral loads compared with their nonadjuvanted counterparts in a SARS-CoV-2 infection model. These data suggest that pADA enhances immune memory and durability and supports further translational studies.

Irreversible Inactivation of SARS-CoV-2 by Lectin Engagement with Two Glycan Clusters on the Spike Protein.

Host cell infection by SARS-CoV-2, similar to that by HIV-1, is driven by a conformationally metastable and highly glycosylated surface entry protein complex, and infection by these viruses has been shown to be inhibited by the mannose-specific lectins cyanovirin-N (CV-N) and griffithsin (GRFT). We discovered in this study that CV-N not only inhibits SARS-CoV-2 infection but also leads to irreversibly inactivated pseudovirus particles. The irreversibility effect was revealed by the observation that pseudoviruses first treated with CV-N and then washed to remove all soluble lectin did not recover infectivity. The infection inhibition of SARS-CoV-2 pseudovirus mutants with single-site glycan mutations in spike suggested that two glycan clusters in S1 are important for both CV-N and GRFT inhibition: one cluster associated with the RBD (receptor binding domain) and the second with the S1/S2 cleavage site. We observed lectin antiviral effects with several SARS-CoV-2 pseudovirus variants, including the recently emerged omicron, as well as a fully infectious coronavirus, therein reflecting the breadth of lectin antiviral function and the potential for pan-coronavirus inactivation. Mechanistically, observations made in this work indicate that multivalent lectin interaction with S1 glycans is likely a driver of the lectin infection inhibition and irreversible inactivation effect and suggest the possibility that lectin inactivation is caused by an irreversible conformational effect on spike. Overall, lectins' irreversible inactivation of SARS-CoV-2, taken with their breadth of function, reflects the therapeutic potential of multivalent lectins targeting the vulnerable metastable spike before host cell encounter.

Debulking SARS-CoV-2 in saliva using angiotensin converting enzyme 2 in chewing gum to decrease oral virus transmission and infection.

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus-trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical-grade plant material was developed to meet FDA requirements. Chewing gum (2 g) containing plant cells expressed CTB-ACE2 up to 17.2 mg ACE2/g dry weight (11.7% leaf protein), have physical characteristics and taste/flavor like conventional gums, and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of lentivirus spike or VSV-spike pseudovirus into Vero/CHO cells when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV-2 virus count in COVID-19 swab/saliva samples by >95% when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared with healthy individuals (2,582 versus 50,126 ΔRFU; 27 versus 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-CoV-2 receptor-binding domain, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus-trapping proteins offers a general affordable strategy to protect patients from most oral virus re-infections through debulking or minimizing transmission to others.

SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed. We have developed multiple serologic assays using newly designed SARS-CoV-2 reagents for detecting the presence of receptor-binding antibodies in sera. The first assay is surface plasmon resonance (SPR) based and can quantitate both antibody binding to the SARS-CoV-2 spike protein and blocking to the Angiotensin-converting enzyme 2 (ACE2) receptor in a single experiment. The second assay is enzyme-linked immunosorbent assay (ELISA) based and can measure competition and blocking of the ACE2 receptor to the SARS-CoV-2 spike protein with antispike antibodies. The assay is highly versatile, and we demonstrate the broad utility of the assay by measuring antibody functionality of sera from small animals and nonhuman primates immunized with an experimental SARS-CoV-2 vaccine. In addition, we employ the assay to measure receptor blocking of sera from SARS-CoV-2-infected patients. The assay is shown to correlate with pseudovirus neutralization titers. This type of rapid, surrogate neutralization diagnostic can be employed widely to help study SARS-CoV-2 infection and assess the efficacy of vaccines.

Structural genomics for drug design against the pathogen Coxiella burnetii.

Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in C. burnetii that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein, C. burnetii dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by in silico screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria.

In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Multivalent in vivo delivery of DNA-encoded bispecific T cell engagers effectively controls heterogeneous GBM tumors and mitigates immune escape.

Glioblastoma multiforme (GBM) is among the most difficult cancers to treat with a 5-year survival rate less than 5%. An immunotherapeutic vaccine approach targeting GBM-specific antigen, EGFRvIII, previously demonstrated important clinical impact. However, immune escape variants were reported in the trial, suggesting that multivalent approaches targeting GBM-associated antigens may be of importance. Here we focused on multivalent in vivo delivery of synthetic DNA-encoded bispecific T cell engagers (DBTEs) targeting two GBM-associated antigens, EGFRvIII and HER2. We designed and optimized an EGFRvIII-DBTE that induced T cell-mediated cytotoxicity against EGFRvIII-expressing tumor cells. In vivo delivery in a single administration of EGFRvIII-DBTE resulted in durable expression over several months in NSG mice and potent tumor control and clearance in both peripheral and orthotopic animal models of GBM. Next, we combined delivery of EGFRvIII-DBTEs with an HER2-targeting DBTE to treat heterogeneous GBM tumors. In vivo delivery of dual DBTEs targeting these two GBM-associated antigens exhibited enhanced tumor control and clearance in a heterogeneous orthotopic GBM challenge, while treatment with single-target DBTE ultimately allowed for tumor escape. These studies support that combined delivery of DBTEs, targeting both EGFRvIII and HER2, can potentially improve outcomes of GBM immunotherapy, and such multivalent approaches deserve additional study.

Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

DNA immunotherapy targeting BARF1 induces potent anti-tumor responses against Epstein-Barr-virus-associated carcinomas.

Latent Epstein-Barr virus (EBV) infection is associated with several types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV malignancies, suggesting that additional targets might be important. BamHI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV+ nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC). BARF1 antigen can transform human epithelial cells in vivo. BARF1-specific antibodies and cytotoxic T cells were detected in some EBV+ NPC patients. However, BARF1 has not been evaluated as an antigen in the context of therapeutic immunization. Its possible importance in this context is unclear. Here, we developed a synthetic-DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and T cell responses in vivo. Immunization with pBARF1 plasmid impacted tumor progression through the induction of CD8+ T cells in novel BARF1+ carcinoma models. Using an in vivo imaging system, we observed that pBARF1-immunized animals rapidly cleared cancer cells. We demonstrated that pBARF1 can induce antigen-specific immune responses that can impact cancer progression. Further study of this immune target is likely important as part of therapeutic approaches for EBV+ malignancies.

In vivo DNA-launched bispecific T cell engager targeting IL-13Rα2 controls tumor growth in an animal model of glioblastoma multiforme.

Glioblastoma is an aggressive tumor with poor survival rates. Bispecific T cell engagers (BTEs) against different cancers are in various stages of clinical development. Toxicity resulting from cytokine release syndrome and the short half-life of BTEs, which necessitates continuous infusion, complicating delivery and increasing costs, are major challenges in the field. Here we describe the development of in vivo DNA-launched BTEs (dBTEs) with highly focused targeting of interleukin-13 receptor α2 (IL-13Rα2), a glioblastoma cell-surface target. We developed 4 BTEs targeting 2 epitopes of IL-13Rα2 and studied how heavy-light chain orientation affects BTE function. The dBTEs induced T cell activation, cytokine production, and tumor cytolysis in the presence of IL-13Rα2+ tumor cells, but we observed unique patterns of immune activation. We found a strong correlation between granzyme B secretion and dBTE-induced cytolysis of specific and nonspecific tumors. We down-selected dBTE PB01-forward based on lower cytokine induction profile and highest activation specificity. In vivo, dBTE PB01-forward demonstrated an improved half-life versus intravenous recombinant BTE delivery. In an orthotopic glioblastoma model, dBTE PB01-forward controlled tumor growth, improving animal survival, supporting the hypothesis that the blood-brain barrier does not affect the function of systemically delivered dBTE. Further study of PB01-forward for targeting glioblastoma and other IL-13Rα2+ cancers is warranted.

Mucosal chemokine adjuvant enhances synDNA vaccine-mediated responses to SARS-CoV-2 and provides heterologous protection in vivo.

The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ)+ CD8+ T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important.

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates.

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2.

Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.

A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions.

Intradermal-delivered DNA vaccine induces durable immunity mediating a reduction in viral load in a rhesus macaque SARS-CoV-2 challenge model.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, has had a dramatic global impact on public health and social and economic infrastructures. Here, we assess the immunogenicity and anamnestic protective efficacy in rhesus macaques of an intradermal (i.d.)-delivered SARS-CoV-2 spike DNA vaccine, INO-4800, currently being evaluated in clinical trials. Vaccination with INO-4800 induced T cell responses and induced spike antigen and RBD binding antibodies with ADCP and ADCD activity. Sera from the animals neutralized both the D614 and G614 SARS-CoV-2 pseudotype viruses. Several months after vaccination, animals were challenged with SARS-CoV-2 resulting in rapid recall of anti-SARS-CoV-2 spike protein T cell and neutralizing antibody responses. These responses were associated with lower viral loads in the lung. These studies support the immune impact of INO-4800 for inducing both humoral and cellular arms of the adaptive immune system, which are likely important for providing durable protection against COVID-19 disease.

DNA vaccines: prime time is now.

Recently newer synthetic DNA vaccines have been rapidly advanced to clinical study and have demonstrated an impressive degree of immune potency and tolerability. Improvements in DNA delivery over prior needle and syringe approaches include jet delivery, gene gun delivery, among others. Among the most effective of these new delivery methods, advanced electroporation (EP), combined with other advances, induces robust humoral and cellular immunity in both preventative as well as therapeutic studies. Advancements in the design of the DNA inserts include leader sequence changes, RNA and codon optimizations, improved insert designs, increased concentrations of DNA, and skin delivery, appear to complement newer delivery strategies. These advances also provide a framework for the in vivo production of synthetic DNA biologics. In this review, we focus on recent studies of synthetic DNA vaccines in the clinic for the prevention or treatment of infectious diseases with a focus on adaptive electroporation for delivery, and briefly summarize novel preclinical data advancing the in vivo delivery of DNA-encoded antibody-like biologics.

Adenosine deaminase-1 enhances germinal center formation and functional antibody responses to HIV-1 Envelope DNA and protein vaccines.

Adenosine deaminase-1 (ADA-1) plays both enzymatic and non-enzymatic roles in regulating immune cell function. Mutations in the ADA1 gene account for 15% of heritable severe-combined immunodeficiencies. We determined previously that ADA1 expression defines and is instrumental for the germinal center follicular helper T cell (TFH) phenotype using in vitro human assays. Herein, we tested whether ADA-1 can be used as an adjuvant to improve vaccine efficacy in vivo. In vitro, ADA-1 induced myeloid dendritic cell (mDC) maturation as measured by increased frequencies of CD40-, CD83-, CD86-, and HLA-DR-positive mDCs. ADA-1 treatment also promoted the secretion of the TFH-polarizing cytokine IL-6 from mDCs. In the context of an HIV-1 envelope (env) DNA vaccine, co-immunization with plasmid-encoded ADA-1 (pADA) enhanced humoral immunity. Animals co-immunized with env DNA and pADA had significantly increased frequencies of TFH cells in their draining lymph nodes and increased HIV-binding IgG in serum. Next, mice were co-immunized with subtype C env gp160 DNA and pADA along with simultaneous immunization with matched gp140 trimeric protein. Mice that received env gp160 DNA, pADA, and gp140 glycoprotein had significantly more heterologous HIV-specific binding IgG in their serum. Furthermore, only these mice had detectable neutralizing antibody responses. These studies support the use of ADA-1 as a vaccine adjuvant to qualitatively enhance germinal center responses and represent a novel application of an existing therapeutic agent that can be quickly translated for clinical use.

Immunogenicity of a DNA vaccine candidate for COVID-19.

The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.

Defensive Driving: Directing HIV-1 Vaccine-Induced Humoral Immunity to the Mucosa with Chemokine Adjuvants.

A myriad of pathogens gain access to the host via the mucosal route; thus, vaccinations that protect against mucosal pathogens are critical. Pathogens such as HIV, HSV, and influenza enter the host at mucosal sites such as the intestinal, urogenital, and respiratory tracts. All currently licensed vaccines mediate protection by inducing the production of antibodies which can limit pathogen replication at the site of infection. Unfortunately, parenteral vaccination rarely induces the production of an antigen-specific antibody at mucosal surfaces and thus relies on transudation of systemically generated antibody to mucosal surfaces to mediate protection. Mucosa-associated lymphoid tissues (MALTs) consist of a complex network of immune organs and tissues that orchestrate the interaction between the host, commensal microbes, and pathogens at these surfaces. This complexity necessitates strict control of the entry and exit of lymphocytes in the MALT. This control is mediated by chemoattractant chemokines or cytokines which recruit immune cells expressing the cognate receptors and adhesion molecules. Exploiting mucosal chemokine trafficking pathways to mobilize specific subsets of lymphocytes to mucosal tissues in the context of vaccination has improved immunogenicity and efficacy in preclinical models. This review describes the novel use of MALT chemokines as vaccine adjuvants. Specific attention will be placed upon the use of such adjuvants to enhance HIV-specific mucosal humoral immunity in the context of prophylactic vaccination.

Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus-Cell Interface.

To address the urgent need for new agents to reduce the global occurrence and spread of AIDS, we investigated the underlying hypothesis that antagonists of the HIV-1 envelope (Env) gp120 protein and the host-cell coreceptor (CoR) protein can be covalently joined into bifunctional synergistic combinations with improved antiviral capabilities. A synthetic protocol was established to covalently combine a CCR5 small-molecule antagonist and a gp120 peptide triazole antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the specific targeting properties of the two separate chimera components and, at the same time, exhibited low to subnanomolar potencies in inhibiting cell infection by different pseudoviruses, which were substantially greater than those of a noncovalent mixture of the individual components. The results demonstrate that targeting the virus-cell interface with a single molecule can result in improved potencies and also the introduction of new phenotypes to the chimeric inhibitor, such as the irreversible inactivation of HIV-1.