Intestinal Interleukin-17 Receptor Signaling Mediates Reciprocal Control of the Gut Microbiota and Autoimmune Inflammation.

Interleukin-17 (IL-17) and IL-17 receptor (IL-17R) signaling are essential for regulating mucosal host defense against many invading pathogens. Commensal bacteria, especially segmented filamentous bacteria (SFB), are a crucial factor that drives T helper 17 (Th17) cell development in the gastrointestinal tract. In this study, we demonstrate that Th17 cells controlled SFB burden. Disruption of IL-17R signaling in the enteric epithelium resulted in SFB dysbiosis due to reduced expression of α-defensins, Pigr, and Nox1. When subjected to experimental autoimmune encephalomyelitis, IL-17R-signaling-deficient mice demonstrated earlier disease onset and worsened severity that was associated with increased intestinal Csf2 expression and elevated systemic GM-CSF cytokine concentrations. Conditional deletion of IL-17R in the enteric epithelium demonstrated that there was a reciprocal relationship between the gut microbiota and enteric IL-17R signaling that controlled dysbiosis, constrained Th17 cell development, and regulated the susceptibility to autoimmune inflammation.

Elastic behavior of model membranes with antimicrobial peptides depends on lipid specificity and d-enantiomers.

In an effort to provide new treatments for the global crisis of bacterial resistance to current antibiotics, we have used a rational approach to design several new antimicrobial peptides (AMPs). The present study focuses on 24-mer WLBU2 and its derivative, D8, with the amino acid sequence, RRWVRRVRRWVRRVVRVVRRWVRR. In D8, all of the valines are the d-enantiomer. We use X-ray low- and wide-angle diffuse scattering data to measure elasticity and lipid chain order. We show a good correlation between in vitro bacterial killing efficiency and both bending and chain order behavior in bacterial lipid membrane mimics; our results suggest that AMP-triggered domain formation could be the mechanism of bacterial killing in both Gram-positive and Gram-negative bacteria. In red blood cell lipid mimics, D8 stiffens and orders the membrane, while WLBU2 softens and disorders it, which correlate with D8's harmless vs. WLBU2's toxic behavior in hemolysis tests. These results suggest that elasticity and chain order behavior can be used to predict mechanisms of bactericidal action and toxicity of new AMPs.

Protective efficacy of centralized and polyvalent envelope immunogens in an attenuated equine lentivirus vaccine.

Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated) equine infectious anemia (EIAV) virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully-functional, replication-competent lentivirus, which when experimentally evaluated, demonstrated broader immunogenicity that does not equate to higher protective efficacy.

Engineered cationic antimicrobial peptide (eCAP) prevents Pseudomonas aeruginosa biofilm growth on airway epithelial cells.

OBJECTIVES: Chronic infections with the opportunistic pathogen Pseudomonas aeruginosa are responsible for the majority of the morbidity and mortality in patients with cystic fibrosis (CF). While P. aeruginosa infections may initially be treated successfully with standard antibiotics, chronic infections typically arise as bacteria transition to a biofilm mode of growth and acquire remarkable antimicrobial resistance. To address the critical need for novel antimicrobial therapeutics that can effectively suppress chronic bacterial infections in challenging physiological environments, such as the CF lung, we have rationally designed a de novo engineered cationic antimicrobial peptide, the 24-residue WLBU2, with broad-spectrum antibacterial activity for pan-drug-resistant P. aeruginosa in liquid culture. In the current study, we tested the hypothesis that WLBU2 also prevents P. aeruginosa biofilm growth. METHODS: Using abiotic and biotic biofilm assays, co-culturing P. aeruginosa with polarized human airway epithelial cells, we examined the ability of WLBU2 to prevent biofilm biogenesis alone and in combination with currently used antibiotics. RESULTS: We observed a dose-dependent reduction in biofilm growth on an abiotic surface and in association with CF airway epithelial cells. WLBU2 prevented P. aeruginosa biofilm formation when co-cultured with mucus-producing primary human CF airway epithelial cells and using CF clinical isolates of P. aeruginosa, even at low pH and high salt conditions that mimic the CF airway. When used in combination, WLBU2 significantly increases killing by the commonly used antibiotics tobramycin, ciprofloxacin, ceftazidime and meropenem. CONCLUSIONS: While other studies have demonstrated the ability of natural and synthetic antimicrobial peptides to prevent abiotic bacterial biofilm formation, the current studies for the first time demonstrate the effective peptide treatment of a biotic bacterial biofilm in a setting similar to the CF airway, and without negative effects on human airway epithelial cells, thus highlighting the unique potential of this engineered cationic antimicrobial peptide for treatment of human respiratory infections.

Unique functional properties of conserved arginine residues in the lentivirus lytic peptide domains of the C-terminal tail of HIV-1 gp41.

A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.

Engineered cationic antimicrobial peptides to overcome multidrug resistance by ESKAPE pathogens.

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings.

Membrane structure correlates to function of LLP2 on the cytoplasmic tail of HIV-1 gp41 protein.

Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (-1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.

Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens.

The emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against both P. aeruginosa and S. aureus under challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens.

C-terminal tail of human immunodeficiency virus gp41: functionally rich and structurally enigmatic.

The human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) pandemic is amongst the most important current worldwide public health threats. While much research has been focused on AIDS vaccines that target the surface viral envelope (Env) protein, including gp120 and the gp41 ectodomain, the C-terminal tail (CTT) of gp41 has received relatively little attention. Despite early studies highlighting the immunogenicity of a particular CTT sequence, the CTT has been classically portrayed as a type I membrane protein limited to functioning in Env trafficking and virion incorporation. Recent studies demonstrate, however, that the Env CTT has other important functions. The CTT has been shown to additionally modulate Env ectodomain structure on the cell and virion surface, affect Env reactivity and viral sensitivity to conformation-dependent neutralizing antibodies, and alter cell-cell and virus-cell fusogenicity of Env. This review provides an overview of the Env structure and function with a particular emphasis on the CTT and recent studies that highlight its functionally rich nature.

Budding of retroviruses utilizing divergent L domains requires nucleocapsid.

We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX(n)L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX(n)L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.

Highly conserved structural properties of the C-terminal tail of HIV-1 gp41 protein despite substantial sequence variation among diverse clades: implications for functions in viral replication.

Although the HIV-1 Env gp120 and gp41 ectodomain have been extensively characterized in terms of structure and function, similar characterizations of the C-terminal tail (CTT) of HIV gp41 remain relatively limited and contradictory. The current study was designed to examine in detail CTT sequence conservation relative to gp120 and the gp41 ectodomain and to examine the conservation of predicted physicochemical and structural properties across a number of divergent HIV clades and groups. Results demonstrate that CTT sequences display intermediate levels of sequence evolution and diversity in comparison to the more diverse gp120 and the more conserved gp41 ectodomain. Despite the relatively high level of CTT sequence variation, the physicochemical properties of the lentivirus lytic peptide domains (LLPs) within the CTT are evidently highly conserved across clades/groups. Additionally, predictions using PEP-FOLD indicate a high level of structural similarity in the LLP regions that was confirmed by circular dichroism measurements of secondary structure of LLP peptides from clades B, C, and group O. Results demonstrate that LLP peptides adopt helical structure in the presence of SDS or trifluoroethanol but are predominantly unstructured in aqueous buffer. Thus, these data for the first time demonstrate strong conservations of characteristic CTT physicochemical and structural properties despite substantial sequence diversity, apparently indicating a delicate balance between evolutionary pressures and the conservation of CTT structure and associated functional roles in virus replication.

The lack of an inherent membrane targeting signal is responsible for the failure of the matrix (M1) protein of influenza A virus to bud into virus-like particles.

The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.

Persistent pneumocystis colonization leads to the development of chronic obstructive pulmonary disease in a nonhuman primate model of AIDS.

Human immunodeficiency virus (HIV)-infected patients are at increased risk for development of pulmonary complications, including chronic obstructive pulmonary disease (COPD). Inflammation associated with subclinical infection has been postulated to promote COPD. Persistence of Pneumocystis is associated with HIV infection and COPD, although a causal relationship has not been established. We used a simian/human immunodeficiency virus model of HIV infection to study pulmonary effects of Pneumocystis colonization. Simian/human immunodeficiency virus-infected/Pneumocystis-colonized monkeys developed progressive obstructive pulmonary disease characterized by increased emphysematous tissue and bronchial-associated lymphoid tissue. Increased levels of T helper type 2 cytokines and proinflammatory mediators in bronchoalveolar lavage fluid coincided with Pneumocystis colonization and a decline in pulmonary function. These results support the concept that an infectious agent contributes to the development of HIV-associated lung disease and suggest that Pneumocystis colonization may be a risk factor for the development of HIV-associated COPD. Furthermore, this model allows examination of early host responses important to disease progression, thus identifying potential therapeutic targets for COPD.

Mass Balance Study of the Engineered Cationic Antimicrobial Peptide, WLBU2, Following a Single Intravenous Dose of 14C-WLBU2 in Mice.

BACKGROUND: To address multidrug resistance, we developed engineered Cationic Antimicrobial Peptides (eCAPs). Lead eCAP WLBU2 displays potent activity against drug-resistant bacteria and effectively treats lethal bacterial infections in mice, reducing bacterial loads to undetectable levels in diverse organs. OBJECTIVE: To support the development of WLBU2, we conducted a mass balance study. METHODS: CD1 mice were administered 10, 15, 20 and 30 mg/kg of QDx5 WLBU2 or a single dose of [14C]-WLBU2 at 15 mg/kg IV. Tolerability, tissue distribution and excretion were evaluated with liquid scintillation and HPLC-radiochromatography. RESULTS: The maximum tolerated dose of WLBU2 is 20 mg/kg IV. We could account for greater than >96% of the radioactivity distributed within mouse tissues at 5 and 15 min. By 24h, only ~40-50% of radioactivity remained in the mice. The greatest % of the dose was present in liver, accounting for ~35% of radioactivity at 5 and 15 min, and ~ 8% of radioactivity remained at 24h. High radioactivity was also present in kidneys, plasma, red blood cells and lungs, while less than 0.2% of radioactivity was present in brain, fat, or skeletal muscle. Urinary and fecal excretion accounted for 12.5 and 2.2% of radioactivity at 24h. CONCLUSION: WLBU2 distributes widely to mouse tissues and is rapidly cleared with a terminal radioactivity half-life of 22 h, a clearance of 27.4 mL/h/kg, and a distribution volume of 0.94 L/kg. At 2-100 µg-eq/g, the concentrations of 14C-WLBU2 appear high enough in the tissues to account for the inhibition of microbial growth.

Relationship of Pneumocystis jiroveci humoral immunity to prevention of colonization and chronic obstructive pulmonary disease in a primate model of HIV infection.

Pulmonary colonization by the opportunistic pathogen Pneumocystis jiroveci is common in HIV(+) subjects and has been associated with development of chronic obstructive pulmonary disease (COPD). Host and environmental factors associated with colonization susceptibility are undefined. Using a simian-human immunodeficiency virus (SHIV) model of HIV infection, the immunologic parameters associated with natural Pneumocystis jiroveci transmission were evaluated. SHIV-infected macaques were exposed to P. jiroveci by cohousing with immunosuppressed, P. jiroveci-colonized macaques in two independent experiments. Serial plasma and bronchoalveolar lavage (BAL) fluid samples were examined for changes in antibody titers to recombinant Pneumocystis-kexin protein (KEX1) and evidence of Pneumocystis colonization by nested PCR of BAL fluid. In experiment 1, 10 of 14 monkeys became Pneumocystis colonized (Pc(+)) by 8 weeks post-SHIV infection, while 4 animals remained Pneumocystis colonization negative (Pc(-)) throughout the study. In experiment 2, 11 of 17 animals became Pneumocystis colonized by 16 weeks post-SHIV infection, while 6 monkeys remained Pc(-). Baseline plasma KEX1-IgG titers were significantly higher in monkeys that remained Pc(-), compared to Pc(+) monkeys, in experiments 1 (P = 0.013) and 2 (P = 0.022). Pc(-) monkeys had greater percentages of Pneumocystis-specific memory B cells after SHIV infection compared to Pc(+) monkeys (P = 0.037). After SHIV infection, Pc(+) monkeys developed progressive obstructive pulmonary disease, whereas Pc(-) monkeys maintained normal lung function throughout the study. These results demonstrate a correlation between the KEX1 humoral response and the prevention of Pneumocystis colonization and obstructive lung disease in the SHIV model. In addition, these results indicate that an effective Pneumocystis-specific memory B-cell response is maintained despite progressive loss of CD4(+) T cells during SHIV infection.

Replication of equine infectious anemia virus in engineered mouse NIH 3T3 cells.

We employed the equine lentivirus equine infectious anemia virus (EIAV) to investigate the cellular restrictions for lentivirus replication in murine NIH 3T3 cells. The results of these studies demonstrate that NIH 3T3 cells expressing the EIAV receptor ELR1 and equine cyclin T1 supported productive replication of EIAV and produced infectious virions at levels similar to those found in a reference permissive equine cell line. The studies presented here demonstrate, for the first time, differential levels of restriction for EIAV and human immunodeficiency virus type 1 (HIV-1) replication in murine cells and suggest that these differences can be exploited to reveal critical virus-cell interactions required for HIV-1 assembly and budding of lentivirus particles.

Human immunodeficiency virus type 1 Vpr: oligomerization is an essential feature for its incorporation into virus particles.

HIV-1 Vpr, a nonstructural viral protein associated with virus particles, has a positive role in the efficient transport of PIC into the nucleus of non-dividing target cells and enhances virus replication in primary T cells. Vpr is a 96 amino acid protein and the structure by NMR shows three helical domains. Vpr has been shown to exist as dimers and higher order oligomers. Considering the multifunctional nature of Vpr, the contribution of distinct helical domains to the dimer/oligomer structure of Vpr and the relevance of this feature to its functions are not clear. To address this, we have utilized molecular modeling approaches to identify putative models of oligomerization. The predicted interface residues were subjected to site-directed mutagenesis and evaluated their role in intermolecular interaction and virion incorporation. The interaction between Vpr molecules was monitored by Bimolecular Fluorescence complementation (BiFC) method. The results show that Vpr forms oligomers in live cells and residues in helical domains play critical roles in oligomerization. Interestingly, Vpr molecules defective in oligomerization also fail to incorporate into the virus particles. Based on the data, we suggest that oligomerization of Vpr is essential for virion incorporation property and may also have a role in the events associated with virus infection.

Engineered Cationic Antimicrobial Peptides (eCAPs) to Combat Multidrug-Resistant Bacteria.

The increasing rate of antibiotic resistance constitutes a global health crisis. Antimicrobial peptides (AMPs) have the property to selectively kill bacteria regardless of resistance to traditional antibiotics. However, several challenges (e.g., reduced activity in the presence of serum and lack of efficacy in vivo) to clinical development need to be overcome. In the last two decades, we have addressed many of those challenges by engineering cationic AMPs de novo for optimization under test conditions that typically inhibit the activities of natural AMPs, including systemic efficacy. We reviewed some of the most promising data of the last two decades in the context of the advancement of the field of helical AMPs toward clinical development.

An EIAV field isolate reveals much higher levels of subtype variability than currently reported for the equine lentivirus family.

BACKGROUND: Equine infectious anemia virus (EIAV), a lentivirus that infects horses, has been utilized as an animal model for the study of HIV. Furthermore, the disease associated with the equine lentivirus poses a significant challenge to veterinary medicine around the world. As with all lentiviruses, EIAV has been shown to have a high propensity for genomic sequence and antigenic variation, especially in its envelope (Env) proteins. Recent studies have demonstrated Env variation to be a major determinant of vaccine efficacy, emphasizing the importance of defining natural variation among field isolates of EIAV. To date, however, published EIAV sequences have been reported only for cell-adapted strains of virus, predominantly derived from a single primary virus isolate, EIAVWyoming (EIAVWY). RESULTS: We present here the first characterization of the Env protein of a natural primary isolate from Pennsylvania (EIAVPA) since the widely utilized and referenced EIAVWY strain. The data demonstrated that the level of EIAVPA Env amino acid sequence variation, approximately 40% as compared to EIAVWY, is much greater than current perceptions or published reports of natural EIAV variation between field isolates. This variation did not appear to give rise to changes in the predicted secondary structure of the proteins. While the EIAVPA Env was serologically cross reactive with the Env proteins of the cell-adapted reference strain, EIAVPV (derivative of EIAVWY), the two variant Envs were shown to lack any cross neutralization by immune serum from horses infected with the respective virus strains. CONCLUSION: Taking into account the significance of serum neutralization to universal vaccine efficacy, these findings are crucial considerations towards successful EIAV vaccine development and the potential inclusion of field isolate Envs in vaccine candidates.

An equine infectious anemia virus variant superinfects cells through novel receptor interactions.

Wild-type strains of equine infectious anemia virus (EIAV) prevent superinfection of previously infected cells. A variant strain of virus that spontaneously arose during passage, EIAV(vMA-1c), can circumvent this mechanism in some cells, such as equine dermis (ED) cells, but not in others, such as equine endothelial cells. EIAV(vMA-1c) superinfection of ED cells results in a buildup of unintegrated viral DNA and rapid killing of the cell monolayer. Here, we examined the mechanism of resistance that is used by EIAV to prevent superinfection and explored the means by which EIAV(vMA-1c) overcomes this restriction. We found that the cellular receptor used by EIAV, equine lentivirus receptor 1 (ELR1), remains on the surface of cells chronically infected with EIAV, suggesting that wild-type EIAV interferes with superinfection by masking ELR1. The addition of soluble wild-type SU protein to the medium during infection blocked infection by wild-type strains of virus, implicating SU as the viral protein responsible for interfering with virion entry into previously infected cells. Additionally, interference of wild-type EIAV binding to ELR1 by the addition of either anti-ELR1 antibodies or the ELR1 ectodomain prevented entry of the wild-type strains of EIAV into two permissive cell populations. Many of these same interference treatments prevented EIAV(vMA-1c) infection of endothelial cells but only modestly affected the ability of EIAV(vMA-1c) to enter and kill previously infected ED cells. These findings indicate that EIAV(vMA-1c) retains the ability to use ELR1 for entry and suggest that this virus can interact with an additional, unidentified receptor to superinfect ED cells.