Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope.

Antigenic similarities between Zika virus (ZIKV) and other flaviviruses pose challenges to the development of virus-specific diagnostic tools and effective vaccines. Starting with a DNA-encoded one-bead-one-compound combinatorial library of 508,032 synthetic, non-natural oligomers, we selected and characterized small molecules that mimic ZIKV epitopes. High-throughput fluorescence-activated cell sorter-based bead screening was used to select molecules that bound IgG from ZIKV-immune but not from dengue-immune sera. Deep sequencing of the DNA from the "Zika-only" beads identified 40 candidate molecular structures. A lead candidate small molecule "CZV1-1" was selected that correctly identifies serum specimens from Zika-experienced patients with good sensitivity and specificity (85.3% and 98.4%, respectively). Binding competition studies of purified anti-CZV1-1 IgG against known ZIKV-specific monoclonal antibodies (mAbs) showed that CZV1-1 mimics a nonlinear, neutralizing conformational epitope in the domain III of the ZIKV envelope. Purified anti-CZV1-1 IgG neutralized infection of ZIKV in cell cultures with potencies comparable to highly specific ZIKV-neutralizing mAbs. This study demonstrates an innovative approach for identification of synthetic non-natural molecular mimics of conformational virus epitopes. Such molecular mimics may have value in the development of accurate diagnostic assays for Zika, as well as for other viruses.

Pathogenic Correlates of Simian Immunodeficiency Virus-Associated B Cell Dysfunction.

We compared and contrasted pathogenic (in pig-tailed macaques [PTMs]) and nonpathogenic (in African green monkeys [AGMs]) SIVsab infections to assess the significance of the B cell dysfunction observed in simian (SIV) and human immunodeficiency virus (HIV) infections. We report that the loss of B cells is specifically associated with the pathogenic SIV infection, while in the natural hosts, in which SIV is nonpathogenic, B cells rapidly increase in both lymph nodes (LNs) and intestine. SIV-associated B cell dysfunction associated with the pathogenic SIV infection is characterized by loss of naive B cells, loss of resting memory B cells due to their redistribution to the gut, increases of the activated B cells and circulating tissue-like memory B cells, and expansion of the B regulatory cells (Bregs). While circulating B cells are virtually restored to preinfection levels during the chronic pathogenic SIV infection, restoration is mainly due to an expansion of the "exhausted," virus-specific B cells, i.e., activated memory cells and tissue-like memory B cells. Despite of the B cell dysfunction, SIV-specific antibody (Ab) production was higher in the PTMs than in AGMs, with the caveat that rapid disease progression in PTMs was strongly associated with lack of anti-SIV Ab. Neutralization titers and the avidity and maturation of immune responses did not differ between pathogenic and nonpathogenic infections, with the exception of the conformational epitope recognition, which evolved from low to high conformations in the natural host. The patterns of humoral immune responses in the natural host are therefore more similar to those observed in HIV-infected subjects, suggesting that natural hosts may be more appropriate for modeling the immunization strategies aimed at preventing HIV disease progression. The numerous differences between the pathogenic and nonpathogenic infections with regard to dynamics of the memory B cell subsets point to their role in the pathogenesis of HIV/SIV infections and suggest that monitoring B cells may be a reliable approach for assessing disease progression.IMPORTANCE We report here that the HIV/SIV-associated B cell dysfunction (defined by loss of total and memory B cells, increased B regulatory cell [Breg] counts, and B cell activation and apoptosis) is specifically associated with pathogenic SIV infection and absent during the course of nonpathogenic SIV infection in natural nonhuman primate hosts. Alterations of the B cell population are not correlated with production of neutralizing antibodies, the levels of which are similar in the two species. Rapid progressive infections are associated with a severe impairment in SIV-specific antibody production. While we did not find major differences in avidity and maturation between the pathogenic and nonpathogenic SIV infections, we identified a major difference in conformational epitope recognition, with the nonpathogenic infection being characterized by an evolution from low to high conformations. B cell dysfunction should be considered in designing immunization strategies aimed at preventing HIV disease progression.

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.

Scavenger receptor function of mouse Fcγ receptor III contributes to progression of atherosclerosis in apolipoprotein E hyperlipidemic mice.

Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis.

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.

Sequential seasonal H1N1 influenza virus infections protect ferrets against novel 2009 H1N1 influenza virus.

Individuals <60 years of age had the lowest incidence of infection, with ~25% of these people having preexisting, cross-reactive antibodies to novel 2009 H1N1 influenza. Many people >60 years old also had preexisting antibodies to novel H1N1. These observations are puzzling because the seasonal H1N1 viruses circulating during the last 60 years were not antigenically similar to novel H1N1. We therefore hypothesized that a sequence of exposures to antigenically different seasonal H1N1 viruses can elicit an antibody response that protects against novel 2009 H1N1. Ferrets were preinfected with seasonal H1N1 viruses and assessed for cross-reactive antibodies to novel H1N1. Serum from infected ferrets was assayed for cross-reactivity to both seasonal and novel 2009 H1N1 strains. These results were compared to those of ferrets that were sequentially infected with H1N1 viruses isolated prior to 1957 or more-recently isolated viruses. Following seroconversion, ferrets were challenged with novel H1N1 influenza virus and assessed for viral titers in the nasal wash, morbidity, and mortality. There was no hemagglutination inhibition (HAI) cross-reactivity in ferrets infected with any single seasonal H1N1 influenza viruses, with limited protection to challenge. However, sequential H1N1 influenza infections reduced the incidence of disease and elicited cross-reactive antibodies to novel H1N1 isolates. The amount and duration of virus shedding and the frequency of transmission following novel H1N1 challenge were reduced. Exposure to multiple seasonal H1N1 influenza viruses, and not to any single H1N1 influenza virus, elicits a breadth of antibodies that neutralize novel H1N1 even though the host was never exposed to the novel H1N1 influenza viruses.

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.

Elicitation of anti-1918 influenza virus immunity early in life prevents morbidity and lower levels of lung infection by 2009 pandemic H1N1 influenza virus in aged mice.

The Spanish influenza virus pandemic of 1918 was responsible for 40 million to 50 million deaths and is antigenically similar to the swine lineage 2009 pandemic influenza virus. Emergence of the 2009 pandemic from swine into humans has raised the possibility that low levels of cross-protective immunity to past shared epitopes could confer protection. In this study, influenza viruslike particles (VLPs) were engineered to express the hemagglutinin (HA) and genes from the 1918 influenza virus to evaluate the duration of cross-protection to the H1N1 pandemic strain by vaccinating young mice (8 to 12 weeks) and then allowing the animals to age to 20 months. This immunity was long lasting, with homologous receptor-blocking antibodies detected throughout the lifespan of vaccinated mice. Furthermore, the 1918 VLPs fully protected aged mice from 2009 pandemic H1N1 virus challenge 16 months after vaccination. Histopathological assessment showed that aged vaccinated mice had significant protection from alveolar infection but less protection of the bronchial tissue than adult vaccinated mice. Additionally, passive transfer of immune serum from aged vaccinated mice resulted in protection from death but not morbidity. This is the first report describing the lifelong duration of cross-reactive immune responses elicited by a 1918 VLP vaccine in a murine model. Importantly, these lifelong immune responses did not result in decreased total viral replication but did prevent infection of the lower respiratory tract. These findings show that immunity acquired early in life can restrict the anatomical location of influenza viral replication, rather than preventing infection, in the aged.

Functional characterization of ferret CCL20 and CCR6 and identification of chemotactic inhibitors.

CCL20 is currently the only known chemokine ligand for the receptor CCR6, and is a mucosal chemokine involved in normal and pathological immune responses. Although nucleotide sequence data are available for ccl20 and ccr6 sequences from multiple species, the ferret ccl20 and ccr6 sequences have not been determined. To increase our understanding of immune function in ferret models of infection and vaccination, we have used RT-PCR to obtain the ferret ccl20 and ccr6 cDNA sequences and functionally characterize the encoded proteins. The open reading frames of both genes were highly conserved across species and mostly closely related to canine sequences. For functional analyses, single cell clones expressing ferret CCR6 were generated, a ferret CCL20/mouse IgG(2a) fusion protein (fCCL20-mIgG(2a)) was produced, and fCCL20 was chemically synthesized. Cell clones expressing ferret CCR6 responded chemotactically to fCCL20-mIgG2a fusion protein and synthetic ferret CCL20. Chemotaxis inhibition studies identified the polyphenol epigallocatechin-3-gallate and the murine γ-herpesvirus 68 M3 protein as inhibitors of fCCL20. Surface plasmon resonance studies revealed that EGCG bound directly to fCCL20. These results provide molecular characterization of previously unreported ferret immune gene sequences and for the first time identify a broad-spectrum small molecule inhibitor of CCL20 and reveal CCL20 as a target for the herpesviral M3 protein.

Epigallocatechin-3-gallate reduces airway inflammation in mice through binding to proinflammatory chemokines and inhibiting inflammatory cell recruitment.

One major activity of chemokines is the recruitment of immune cells to sites of infection and inflammation. CD4(+) Th1 cells play critical roles in host defense against pathogens and in the pathogenesis of many immune-mediated diseases. It was reported that epigallocatechin-3-gallate (EGCG) exhibits anti-inflammatory properties, but the mechanisms have not been completely defined. In this study, we found that EGCG markedly decreased recruitment of murine OVA-specific Th1 cells and other inflammatory cells into the airways in a Th1 adoptive-transfer mouse model. In vitro analysis revealed that EGCG inhibited CXCR3 ligand-driven chemotaxis of murine and human cells. Surface plasmon resonance studies revealed that EGCG bound directly to chemokines CXCL9, CXCL10, and CXCL11. These results indicated that one anti-inflammatory mechanism of EGCG is binding of proinflammatory chemokines and limiting their biological activities. These findings support further development of EGCG as a potent therapeutic for inflammatory diseases.

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.

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.

Envelope determinants of equine infectious anemia virus vaccine protection and the effects of sequence variation on immune recognition.

A highly effective attenuated equine infectious anemia virus (EIAV) vaccine (EIAV(D9)) capable of protecting 100% of horses from disease induced by a homologous Env challenge strain (EIAV(PV)) was recently tested in ponies to determine the level of protection against divergent Env challenge strains (J. K. Craigo, B. S. Zhang, S. Barnes, T. L. Tagmyer, S. J. Cook, C. J. Issel, and R. C. Montelaro, Proc. Natl. Acad. Sci. USA 104:15105-15110, 2007). An inverse correlation between challenge strain Env variation and vaccine protection from disease was observed. Given the striking differences in protective immunity, we hypothesized that analysis of the humoral and cellular immune responses to the Env protein could reveal potential determinants of vaccine protection. Neutralization activity against the homologous Env or challenge strain-specific Env in immune sera from the vaccinated ponies did not correlate with protection from disease. Cellular analysis with Env peptide pools did not reveal an association with vaccine protection from disease. However, when individual vaccine-specific Env peptides were utilized, eight cytotoxic-T-lymphocyte (CTL) peptides were found to associate closely with vaccine protection. One of these peptides also yielded the only lymphoproliferative response associated with protective immunity. The identified peptides spanned both variable and conserved regions of gp90. Amino acid divergence within the principal neutralization domain and the identified peptides profoundly affected immune recognition, as illustrated by the inability to detect cross-reactive neutralizing antibodies and the observation that certain peptide-specific CTL responses were altered. In addition to identifying potential Env determinants of EIAV vaccine efficacy and demonstrating the profound effects of defined Env variation on immune recognition, these data also illustrate the sensitivity offered by individual peptides compared to peptide pools in measuring cellular immune responses in lentiviral vaccine trials.

Characterization of an equine macrophage cell line: application to studies of EIAV infection.

EIAV is a monocyte/macrophage tropic virus. To date, even though EIAV has been under investigation for numerous years, very few details have been elucidated about EIAV/macrophage interactions. This is largely due to the absence of an equine macrophage cell line that would support viral replication. Herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (EML) cell line with the functional and immunophenotype characteristics of differentiated equine monocyte derived macrophage(s) (eMDM(s)). These cells possess strong non-specific esterase (NSE) activity, are able to phagocytose fluorescent bioparticles, and produce nitrites in response to LPS. The EML-3C cell line expresses the EIAV receptor for cellular entry (ELR1) and supports replication of the virulent EIAV(PV) biological clone. Thus, EML-3C cells provide a useful cell line possessing equine macrophage related properties for the growth and study of EIAV infection as well as of other equine macrophage tropic viruses.

Characterization of the gp120 of circulating HIV type 1 in a group of infected Chinese blood/plasma donors with asymptomatic and symptomatic stages of the disease.

The C2-V5 regions of the HIV envelope gene were cloned and sequenced from DNA samples of four asymptomatic and five AIDS patients from a cohort of HIV-1-infected Chinese plasma and blood donors. Sequence analysis revealed that regardless of the stage of disease, all the patient's HIV-1 belonged to either clade B' or circulating recombinant form 15 (CRF15) subtype. However, since these blood donors were infected at a time when circulating CRF15 was not known in that region, it is likely these HIV-1 types belong to clade B'. However, the C2-V5 sequences from AIDS patients clustered differently in the phylogenetic tree than those present in asymptomatic patients and showed a significantly higher divergence and lower diversity compared to those from asymptomatic patients. In addition, more syncytia-inducing viral genotypes were present in AIDS patients than in asymptomatic patients. These results contribute to a better understanding of the pathogenesis of circulating HIV-1 in infected Chinese plasma and blood donors, warrant additional investigation of the possible presence of CRF15 in Chinese blood donors, and may have important implications in the future design of therapeutic strategies for treating these infected patients.

Comparative functional properties of engineered cationic antimicrobial peptides consisting exclusively of tryptophan and either lysine or arginine.

We previously reported a series of de novo engineered cationic antibiotic peptides (eCAPs) consisting exclusively of arginine and tryptophan (WR) that display potent activity against diverse multidrug-resistant (MDR) bacterial strains. In this study, we sought to examine the influence of arginine compared to lysine on antibacterial properties by direct comparison of the WR peptides (8-18 residues) with a parallel series of engineered peptides containing only lysine and tryptophan. WR and WK series were compared for antibacterial activity by bacterial killing and growth inhibition assays and for mechanism of peptide-bacteria interactions by surface plasmon resonance and flow cytometry. Mammalian cytotoxicity was also assessed by flow cytometry, haemolytic and tetrazolium-based assays. The shortest arginine-containing peptides (8 and 10 mers) displayed a statistically significant increase in activity compared to the analogous lysine-containing peptides. The WR and WK peptides achieved maximum antibacterial activity at the 12-mer peptide (WK12 or WR12). Further examination of antibacterial mechanisms of the optimally active 12-mer peptides using surface plasmon resonance and flow cytometry demonstrates stronger interactions with Pseudomonasaeruginosa, greater membrane permeabilizing activity, and lower inhibitory effects of divalent cations on activity and membrane permeabilization properties of WR12 compared to WK12 (P < 0.05). Importantly, WK12 and WR12 displayed similar negligible haemolytic and cytotoxic effects at peptide concentrations up to ten times the MIC or 20 times the minimum bactericidal concentration. Thus, arginine, compared to lysine, can indeed yield enhanced antibacterial activity to minimize the required length to achieve functional antimicrobial peptides.

Subcompartmentalization of HIV-1 quasispecies between seminal cells and seminal plasma indicates their origin in distinct genital tissues.

The mononuclear cells and plasma components of semen from HIV-infected subjects have been shown to contain HIV-1. However, there is very little information as to whether distinct HIV-1 population are present in these two seminal compartments or as to their tissue of origin. Phylogenetic analysis of nucleotide sequences of the C2-V5 region of the HIV-1 gp120 from HIV-1 RNA isolated from seminal cells and seminal plasma of five subjects indicates that the HIV-1 population derived from seminal plasma was distinct from that present in seminal cells. Such subcompartmentalization of HIV-1 between seminal cells and seminal plasma was detected as early as 3 months after seroconversion and persisted up to 38 months following seroconversion. Furthermore, comparison of HIV-1 sequences between testis and prostate tissues showed distinct HIV-1 populations in these tissue compartments. In situ real-time (Taqman) PCR analysis of prostate and testis tissues indicated that T lymphocytes were the predominant cells infected with HIV-1 in both of these tissues. Since seminal plasma is derived from prostate and most of the seminal cells originate from the rete testis and epididymis, these results are consistent with the idea that HIV-1 in seminal plasma is derived from the prostate, while HIV-1-infected cells in semen originate mostly from the rete testis and epididymis. These findings provide for the first time evidence of subcompartmentalization of HIV-1 in male genital organs and suggest that intervention strategies such as vasectomy may not prevent sexual transmission.

Persistent HIV type 1 infection in semen and blood compartments in patients after long-term potent antiretroviral therapy.

HIV-1 RNA levels in semen and blood compartments decrease below detection limits during highly active antiretroviral therapy. Despite these therapeutic effects, it is clear that persistent, latent HIV-1 reservoirs are capable of rebounding in the absence of drug treatment or by evolution of escape mutants remain. The current study was designed to examine the presence of latent virus in semen and blood compartments and its evolution following potent combination therapy with indinavir (protease inhibitor) and efavirenz [nonnucleoside reverse transcriptase (RT) inhibitor]. Using an ultrasensitive in situ hybridization assay HIV-1 mRNA was detected in cultured seminal and blood mononuclear cells in all patients up to 1789 days posttherapy. Higher levels of HIV-1 mRNA were consistently detected in seminal mononuclear cells as compared to peripheral blood mononuclear cells (PBMC) in all time points analyzed posttherapy. Analysis of viral RNA from cultured PBMC before and after therapy displayed no evidence of therapy-induced drug resistance in the viral polymerase gene in the majority of patients. However, distinct envelope populations were detected in these viral RNA populations following therapy, indicating possible selection of quasispecies. The observed ongoing replication and evolution in the PBMC viral envelope sequences likely occurred in the seminal compartment HIV populations, given that the seminal cells showed the ability to express HIV-1 mRNA following cultivation. This together with our previous studies (Gupta P, et al.: J Infect Dis 2000;182:79-87) suggest that the genital and blood compartments likely serve as distinct reservoirs harboring latent HIV-1 during prolonged drug therapy.

Epitope shifting of gp90-specific cellular immune responses in EIAV-infected ponies.

Unlike other lentiviruses, EIAV replication can be controlled in most infected horses leading to an inapparent carrier state free of overt clinical signs which lasts for many years. While the resolution of the initial infection is correlated with the appearance of virus specific cellular immune responses, the precise immune mechanisms responsible for control of the infection are not yet identified. Since the virus undergoes rapid mutation following infection, the immune response must also adapt to meet this challenge. We hypothesize that this adaptation involves peptide-specific recognition shifting from immunodominant variable determinants to conserved immunorecessive determinants following EIAV infection. Forty-four peptides, spanning the entire surface unit protein (gp90) of EIAV, were used to monitor peptide-specific T cell responses in vivo over a six-month period following infection. Peptides were injected intradermally and punch biopsies were collected for real-time PCR analysis to monitor the cellular peptide-specific immune responses in vivo. Similar to the CMI response to HIV infection, peptide-specific T cell recognition patterns changed over time. Early post infection (1 month), immune responses were directed to the peptides in the carboxyl-terminus variable region. By six months post infection, the peptide recognition spanned the entire gp90 sequence. These results indicate that peptide recognition broadens during EIAV infection.