The Glycan Hole Area of HIV-1 Envelope Trimers Contributes Prominently to the Induction of Autologous Neutralization.

The human immunodeficiency virus type 1 (HIV-1) trimeric envelope glycoprotein (Env) is heavily glycosylated, creating a dense glycan shield that protects the underlying peptidic surface from antibody recognition. The absence of conserved glycans, due to missing potential N-linked glycosylation sites (PNGS), can result in strain-specific, autologous neutralizing antibody (NAb) responses. Here, we sought to gain a deeper understanding of the autologous neutralization by introducing holes in the otherwise dense glycan shields of the AMC011 and AMC016 SOSIP trimers. Specifically, when we knocked out the N130 and N289 glycans, which are absent from the well-characterized B41 SOSIP trimer, we observed stronger autologous NAb responses. We also analyzed the highly variable NAb responses induced in rabbits by diverse SOSIP trimers from subtypes A, B, and C. Statistical analysis, using linear regression, revealed that the cumulative area exposed on a trimer by glycan holes correlates with the magnitude of the autologous NAb response. IMPORTANCE Forty years after the first description of HIV-1, the search for a protective vaccine is still ongoing. The sole target for antibodies that can neutralize the virus are the trimeric envelope glycoproteins (Envs) located on the viral surface. The glycoprotein surface is covered with glycans that shield off the underlying protein components from recognition by the immune system. However, the Env trimers of some viral strains have holes in the glycan shield. Immunized animals developed antibodies against such glycan holes. These antibodies are generally strain specific. Here, we sought to gain a deeper understanding of what drives these specific immune responses. First, we show that strain-specific neutralizing antibody responses can be increased by creating artificial holes in the glycan shield. Second, when studying a diverse set of Env trimers with different characteristics, we found that the surface area of the glycan holes contributes prominently to the induction of strain-specific neutralizing antibodies.

A novel anti-inflammatory treatment for bradykinin-induced sore throat or pharyngitis.

BACKGROUND: Often thought of as a minor health concern, sore throat or pharyngitis is an important public health issue. It is one of the most common symptoms of upper respiratory diseases including COVID-19 and is a leading cause of physician visits and antibiotic prescriptions. However, few over-the-counter medications are proven to heal sore throat inflammation. METHODS: Adenocarcinomic human alveolar basal epithelial cells (A549 cells) and three dimensional organotypic human respiratory tissues were used to study inflammation and various treatment effects on respiratory epithelia. The cells and tissues were studied both in the presence and absence of bradykinin, one of the first inflammatory mediators of pharyngitis. Inflammation was measured by analyzing the levels of prostaglandin E2 (PGE2), interleukin 8 (IL-8), and leukotriene B4 (LTB4), transepithelial electrical resistance (TEER), and lactate dehydrogenase (LDH) release. Tissue morphology was analyzed by immunohistochemistry. RESULTS: In studying pharyngitis using organotypic human respiratory tissue stimulated with bradykinin, we saw an increase in PGE2 and interleukin-8 (IL-8) in response to bradykinin. Acetyl salicylic acid (ASA), a nonspecific COX inhibitor, was able to mitigate a bradykinin-induced increase in PGE2 in our studies. However, ASA was inflammatory above its therapeutic window, increasing the levels of PGE2 and IL-8 above those seen with bradykinin stimulation alone. We describe a novel, scientifically validated treatment for sore throat, that contains a low dose of ASA and other anti-inflammatory ingredients. CONCLUSION: This study elucidates the complex mechanisms involved in healing pharyngitis, an inflammatory condition of the upper respiratory epithelia. An ASA-based formula (Biovanta) mitigated bradykinin-induced inflammation more strongly than ASA alone in organotypic human respiratory tissues. Surprisingly, we found that many of the most common over the counter sore throat therapies exacerbate inflammation and IL-8 in organotypic human respiratory tissues, suggesting these common treatments may increase the likelihood of further respiratory complications.

Capturing the inherent structural dynamics of the HIV-1 envelope glycoprotein fusion peptide.

The N-terminal fusion peptide (FP) of the human immunodeficiency virus (HIV)-1 envelope glycoprotein (Env) gp41 subunit plays a critical role in cell entry. However, capturing the structural flexibility in the unbound FP is challenging in the native Env trimer. Here, FP conformational isomerism is observed in two crystal structures of a soluble clade B transmitted/founder virus B41 SOSIP.664 Env with broadly neutralizing antibodies (bNAbs) PGT124 and 35O22 to aid in crystallization and that are not specific for binding to the FP. Large rearrangements in the FP and fusion peptide proximal region occur around M530, which remains anchored in the tryptophan clasp (gp41 W623, W628, W631) in the B41 Env prefusion state. Further, we redesigned the FP at position 518 to reinstate the bNAb VRC34.01 epitope. These findings provide further structural evidence for the dynamic nature of the FP and how a bNAb epitope can be restored during vaccine design.

Closing and Opening Holes in the Glycan Shield of HIV-1 Envelope Glycoprotein SOSIP Trimers Can Redirect the Neutralizing Antibody Response to the Newly Unmasked Epitopes.

In HIV-1 vaccine research, native-like, soluble envelope glycoprotein SOSIP trimers are widely used for immunizing animals. The epitopes of autologous neutralizing antibodies (NAbs) induced by the BG505 and B41 SOSIP trimers in rabbits and macaques have been mapped to a few holes in the glycan shields that cover most of the protein surfaces. For BG505 trimers, the dominant autologous NAb epitope in rabbits involves residues that line a cavity caused by the absence of a glycan at residue 241. Here, we blocked this epitope in BG505 SOSIPv4.1 trimer immunogens by knocking in an N-linked glycan at residue 241. We then opened holes elsewhere on the trimer by knocking out single N-linked glycans at residues 197, 234, 276, 332, and 355 and found that NAb responses induced by the 241-glycan-bearing BG505 trimers were frequently redirected to the newly opened sites. The strongest evidence for redirection of the NAb response to neoepitopes, through the opening and closing of glycan holes, was obtained from trimer immunogen groups with the highest occupancy of the N241 site. We also attempted to knock in the N289-glycan to block the sole autologous NAb epitope on the B41 SOSIP.v4.1 trimer. Although a retrospective analysis showed that the new N289-glycan site was substantially underoccupied, we found some evidence for redirection of the NAb response to a neoepitope when this site was knocked in and the N356-glycan site knocked out. In neither study, however, was redirection associated with increased neutralization of heterologous tier 2 viruses.IMPORTANCE Engineered SOSIP trimers mimic envelope-glycoprotein spikes, which stud the surface of HIV-1 particles and mediate viral entry into cells. When used for immunizing test animals, they elicit antibodies that neutralize resistant sequence-matched HIV-1 isolates. These neutralizing antibodies recognize epitopes in holes in the glycan shield that covers the trimer. Here, we added glycans to block the most immunogenic neutralization epitopes on BG505 and B41 SOSIP trimers. In addition, we removed selected other glycans to open new holes that might expose new immunogenic epitopes. We immunized rabbits with the various glycan-modified trimers and then dissected the specificities of the antibody responses. Thus, in principle, the antibody response might be diverted from one site to a more cross-reactive one, which would help in the induction of broadly neutralizing antibodies by HIV-1 vaccines based on envelope glycoproteins.

Epitopes for neutralizing antibodies induced by HIV-1 envelope glycoprotein BG505 SOSIP trimers in rabbits and macaques.

The native-like, soluble SOSIP.664 trimer based on the BG505 clade A env gene of HIV-1 is immunogenic in various animal species, of which the most studied are rabbits and rhesus macaques. The trimer induces autologous neutralizing antibodies (NAbs) consistently but at a wide range of titers and with incompletely determined specificities. A precise delineation of immunogenic neutralization epitopes on native-like trimers could help strategies to extend the NAb response to heterologous HIV-1 strains. One autologous NAb epitope on the BG505 Env trimer is known to involve residues lining a hole in the glycan shield that is blocked by adding a glycan at either residue 241 or 289. This glycan-hole epitope accounts for the NAb response of most trimer-immunized rabbits but not for that of a substantial subset. Here, we have used a large panel of mutant BG505 Env-pseudotyped viruses to define additional sites. A frequently immunogenic epitope in rabbits is blocked by adding a glycan at residue 465 near the junction of the gp120 V5 loop and ß24 strand and is influenced by amino-acid changes in the structurally nearby C3 region. We name this new site the "C3/465 epitope". Of note is that the C3 region was under selection pressure in the infected infant from whom the BG505 virus was isolated. A third NAb epitope is located in the V1 region of gp120, although it is rarely immunogenic. In macaques, NAb responses induced by BG505 SOSIP trimers are more often directed at the C3/465 epitope than the 241/289-glycan hole.

Improving the Immunogenicity of Native-like HIV-1 Envelope Trimers by Hyperstabilization.

The production of native-like recombinant versions of the HIV-1 envelope glycoprotein (Env) trimer requires overcoming the natural flexibility and instability of the complex. The engineered BG505 SOSIP.664 trimer mimics the structure and antigenicity of native Env. Here, we describe how the introduction of new disulfide bonds between the glycoprotein (gp)120 and gp41 subunits of SOSIP trimers of the BG505 and other genotypes improves their stability and antigenicity, reduces their conformational flexibility, and helps maintain them in the unliganded conformation. The resulting next-generation SOSIP.v5 trimers induce strong autologous tier-2 neutralizing antibody (NAb) responses in rabbits. In addition, the BG505 SOSIP.v6 trimers induced weak heterologous NAb responses against a subset of tier-2 viruses that were not elicited by the prototype BG505 SOSIP.664. These stabilization methods can be applied to trimers from multiple genotypes as components of multivalent vaccines aimed at inducing broadly NAbs (bNAbs).

Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike.

For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7 Å and 3.6 Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6 Å, 5.2 Å and 7.4 Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4-17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes.

Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes.IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.

Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C.

We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses.

Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes.

The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.

Design and structure of two HIV-1 clade C SOSIP.664 trimers that increase the arsenal of native-like Env immunogens.

A key challenge in the quest toward an HIV-1 vaccine is design of immunogens that can generate a broadly neutralizing antibody (bnAb) response against the enormous sequence diversity of the HIV-1 envelope glycoprotein (Env). We previously demonstrated that a recombinant, soluble, fully cleaved SOSIP.664 trimer based on the clade A BG505 sequence is a faithful antigenic and structural mimic of the native trimer in its prefusion conformation. Here, we sought clade C native-like trimers with comparable properties. We identified DU422 and ZM197M SOSIP.664 trimers as being appropriately thermostable (Tm of 63.4 °C and 62.7 °C, respectively) and predominantly native-like, as determined by negative-stain electron microscopy (EM). Size exclusion chromatography, ELISA, and surface plasmon resonance further showed that these trimers properly display epitopes for all of the major bnAb classes, including quaternary-dependent, trimer-apex (e.g., PGT145) and gp120/gp41 interface (e.g., PGT151) epitopes. A cryo-EM reconstruction of the ZM197M SOSIP.664 trimer complexed with VRC01 Fab against the CD4 binding site at subnanometer resolution revealed a striking overall similarity to its BG505 counterpart with expected local conformational differences in the gp120 V1, V2, and V4 loops. These stable clade C trimers contribute additional diversity to the pool of native-like Env immunogens as key components of strategies to induce bnAbs to HIV-1.

HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.

A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene.

UNLABELLED: Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies. IMPORTANCE: The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.

Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins.

UNLABELLED: We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505 env gene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds. IMPORTANCE: It is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface.

The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 µg ml(-1). The median IC50 of neutralized viruses was 0.033 µg ml(-1), among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

A potent combination microbicide that targets SHIV-RT, HSV-2 and HPV.

Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p = 0.0187) infection of mice when 10(6) pfu HSV-2 were applied immediately after vaginal challenge and also when 5×10(3) pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×10(6) HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use.

A modified zinc acetate gel, a potential nonantiretroviral microbicide, is safe and effective against simian-human immunodeficiency virus and herpes simplex virus 2 infection in vivo.

We previously showed that a prototype gel comprising zinc acetate (ZA) in carrageenan (CG) protected mice against vaginal and rectal herpes simplex virus 2 (HSV-2) challenge as well as macaques against vaginal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) challenge. In this work, we modified buffers and cosolvents to obtain a stable, nearly iso-osmolal formulation and evaluated its safety and efficacy against SHIV-RT and HSV-2. In vitro toxicity to lactobacilli and Candida albicans was determined. Macaques were given daily doses of ZA and CG (ZA/CG) or CG alone vaginally for 14 days and challenged with SHIV-RT 24 h later. Mice were challenged vaginally or rectally with HSV-2 immediately after a single gel treatment to measure efficacy or vaginally 12 h after daily gel treatment for 7 days to evaluate the gel's impact on susceptibility to HSV-2 infection. The modified ZA/CG neither affected the viability of lactobacilli or C. albicans nor enhanced vaginal HSV-2 infection after daily ZA/CG treatment. Vaginal SHIV-RT infection of macaques was reduced by 66% (P = 0.006) when macaques were challenged 24 h after the last dose of gel. We observed 60% to 80% uninfected mice after vaginal (P < 0.0001) and rectal (P = 0.008) high-dose HSV-2 challenge. The modified ZA/CG gel is safe and effective in animal models and represents a potential candidate to limit the transmission of HIV and HSV-2.

Conformation-dependent recognition of HIV gp120 by designed ankyrin repeat proteins provides access to novel HIV entry inhibitors.

Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

Simian immunodeficiency virus interactions with macaque dendritic cells.

This chapter summarizes advances in the following areas: (1) dendritic cell (DC)-mediated simian immunodeficiency virus (SIV) transmission, (2) role of DCs in innate and adaptive immunity against SIV, and (3) approaches to harness DC function to induce anti-SIV responses. The nonhuman primate (NHP) model of human immunodeficiency virus (HIV) infection in rhesus macaques and other Asian NHP species is highly relevant to advance the understanding of virus-host interactions critical for transmission and disease pathogenesis. HIV infection is associated with changes in frequency, phenotype, and function of the two principal subsets of DCs, myeloid DCs and plasmacytoid DCs. DC biology during pathogenic SIV infection is strikingly similar to that observed in HIV-infected patients. The NHP models provide an opportunity to dissect the requirements for DC-driven SIV infection and to understand how SIV distorts the DC system to its advantage. Furthermore, the SIV model of mucosal transmission enables the study of the earliest events of infection at the portal of entry that cannot be studied in humans, and, importantly, the involvement of DCs. Nonpathogenic infection in African NHP hosts allows investigations into the role of DCs in disease control. Understanding how DCs are altered during SIV infection is critical to the design of therapeutic and preventative strategies against HIV.

In vivo binding and retention of CD4-specific DARPin 57.2 in macaques.

BACKGROUND: The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4(+) cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4(+) cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4(+) cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4(+) cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology.