Implications of the 375W mutation for HIV-1 tropism and vaccine development.

Understanding how different amino acids affect the HIV-1 envelope (Env) trimer will greatly help the design and development of vaccines that induce broadly neutralizing antibodies (bnAbs). A tryptophan residue at position 375 that opens the CD4 binding site without modifying the trimer apex was identified using our saturation mutagenesis strategy. 375W was introduced into a large panel of 27 transmitted/founder, acute stage, chronic infection, and AIDS macrophage-tropic and non-macrophage-tropic primary envelopes from different clades (A, B, C, D, and G) as well as complex and circulating recombinants. We evaluated soluble CD4 and monoclonal antibody neutralization of WT and mutant Envs together with macrophage infection. The 375W substitution increased sensitivity to soluble CD4 in all 27 Envs and macrophage infection in many Envs including an X4 variant. Importantly, 375W did not impair or abrogate neutralization by potent bnAbs. Variants that were already highly macrophage tropic were compromised for macrophage tropism, indicating that other structural factors are involved. Of note, we observed a macrophage-tropic (clade G) and intermediate macrophage-tropic (clades C and D) primary Envs from the blood and not from the central nervous system (CNS), indicating that such variants could be released from the brain or evolve outside the CNS. Our data also indicate that "intermediate" macrophage-tropic variants should belong to a new class of HIV-1 tropism. These Envs infected macrophages more efficiently than non-macrophage-tropic variants without reaching the high levels of macrophage-tropic brain variants. In summary, we show that 375W is ideal for inclusion into HIV-1 vaccines, increasing Env binding to CD4 for widely diverse Envs from different clades and disease stages.IMPORTANCESubstitutions exposing the CD4 binding site (CD4bs) on HIV-1 trimers but still occluding non-neutralizing, immunogenic epitopes are desirable to develop HIV-1 vaccines. If such substitutions induce similar structural changes in trimers across diverse clades, they could be exploited for the development of multi-clade envelope (Env) vaccines. We show that the 375W substitution increases CD4 affinity for envelopes of all clades, circulating recombinant forms, and complex Envs tested, independent of disease stage. Clade B and C Envs with an exposed CD4bs were described for macrophage-tropic strains from the central nervous system (CNS). Here, we show that intermediate (clades C and D) and macrophage-tropic (clade G) envelopes can be detected outside the CNS. Vaccines targeting the CD4bs will be particularly effective against such strains and CNS disease.

HIV-1 R5 Macrophage-Tropic Envelope Glycoprotein Trimers Bind CD4 with High Affinity, while the CD4 Binding Site on Non-macrophage-tropic, T-Tropic R5 Envelopes Is Occluded.

HIV-1 R5 variants exploit CCR5 as a coreceptor to infect both T cells and macrophages. R5 viruses that are transmitted or derived from immune tissue and peripheral blood are mainly inefficient at mediating infection of macrophages. In contrast, highly macrophage-tropic (mac-tropic) R5 viruses predominate in brain tissue and can be detected in cerebrospinal fluid but are infrequent in immune tissue or blood even in late disease. These mac-tropic R5 variants carry envelope glycoproteins (Envs) adapted to exploit low levels of CD4 on macrophages to induce infection. However, it is unclear whether this adaptation is conferred by an increased affinity of the Env trimer for CD4 or is mediated by postbinding structural rearrangements in the trimer that enhance the exposure of the coreceptor binding site and facilitate events leading to fusion and virus entry. In this study, we investigated CD4 binding to mac-tropic and non-mac-tropic Env trimers and showed that CD4-IgG binds efficiently to mac-tropic R5 Env trimers, while binding to non-mac-tropic trimers was undetectable. Our data indicated that the CD4 binding site (CD4bs) is highly occluded on Env trimers of non-mac-tropic R5 viruses. Such viruses may therefore infect T cells via viral synapses where Env and CD4 become highly concentrated. This environment will enable high-avidity interactions that overcome extremely low Env-CD4 affinities.IMPORTANCE HIV R5 variants bind to CD4 and CCR5 receptors on T cells and macrophages to initiate infection. Transmitted HIV variants infect T cells but not macrophages, and these viral strains persist in immune tissue even in late disease. Here we show that the binding site for CD4 present on HIV's envelope protein is occluded on viruses replicating in immune tissue. This occlusion likely prevents antibody binding to this site and neutralization of the virus, but it makes it difficult for virus-CD4 interactions to occur. Such viruses probably pass from T cell to T cell via cell contacts where CD4 is highly concentrated and allows infection via inefficient envelope-CD4 binding. Our data are highly relevant for vaccines that aim to induce antibodies targeting the CD4 binding site on the envelope protein.

Delineating CD4 dependency of HIV-1: Adaptation to infect low level CD4 expressing target cells widens cellular tropism but severely impacts on envelope functionality.

A hallmark of HIV-1 infection is the continuously declining number of the virus' predominant target cells, activated CD4+ T cells. With diminishing CD4+ T cell levels, the capacity to utilize alternate cell types and receptors, including cells that express low CD4 receptor levels such as macrophages, thus becomes crucial. To explore evolutionary paths that allow HIV-1 to acquire a wider host cell range by infecting cells with lower CD4 levels, we dissected the evolution of the envelope-CD4 interaction under in vitro culture conditions that mimicked the decline of CD4high target cells, using a prototypic subtype B, R5-tropic strain. Adaptation to CD4low targets proved to severely alter envelope functions including trimer opening as indicated by a higher affinity to CD4 and loss in shielding against neutralizing antibodies. We observed a strikingly decreased infectivity on CD4high target cells, but sustained infectivity on CD4low targets, including macrophages. Intriguingly, the adaptation to CD4low targets altered the kinetic of the entry process, leading to rapid CD4 engagement and an extended transition time between CD4 and CCR5 binding during entry. This phenotype was also observed for certain central nervous system (CNS) derived macrophage-tropic viruses, highlighting that the functional perturbation we defined upon in vitro adaptation to CD4low targets occurs in vivo. Collectively, our findings suggest that CD4low adapted envelopes may exhibit severe deficiencies in entry fitness and shielding early in their evolution. Considering this, adaptation to CD4low targets may preferentially occur in a sheltered and immune-privileged environment such as the CNS to allow fitness restoring compensatory mutations to occur.

Saturation Mutagenesis of the HIV-1 Envelope CD4 Binding Loop Reveals Residues Controlling Distinct Trimer Conformations.

The conformation of HIV-1 envelope (Env) glycoprotein trimers is key in ensuring protection against waves of neutralizing antibodies generated during infection, while maintaining sufficient exposure of the CD4 binding site (CD4bs) for viral entry. The CD4 binding loop on Env is an early contact site for CD4 while penetration of a proximal cavity by CD4 triggers Env conformational changes for entry. The role of residues in the CD4 binding loop in regulating the conformation of the trimer and trimer association domain (TAD) was investigated using a novel saturation mutagenesis approach. Single mutations identified, resulted in distinct trimer conformations affecting CD4bs exposure, the glycan shield and the TAD across diverse HIV-1 clades. Importantly, mutations that improve access to the CD4bs without exposing the immunodominant V3 loop were identified. The different trimer conformations identified will affect the specificity and breadth of nabs elicited in vivo and are important to consider in design of Env immunogens for vaccines.

Efficiency of bridging-sheet recruitment explains HIV-1 R5 envelope glycoprotein sensitivity to soluble CD4 and macrophage tropism.

HIV-1 R5 viruses vary extensively in their capacity to infect macrophages. R5 viruses that confer efficient infection of macrophages are able to exploit low levels of CD4 for infection and predominate in brain tissue, where macrophages are a major target for infection. HIV-1 R5 founder viruses that are transmitted were reported to be non-macrophage-tropic. Here, we investigated the sensitivities of macrophage-tropic and non-macrophage-tropic R5 envelopes to neutralizing antibodies. We observed striking differences in the sensitivities of Env(+) pseudovirions to soluble CD4 (sCD4) and to neutralizing monoclonal antibodies (MAbs) that target the CD4 binding site. Macrophage-tropic R5 Envs were sensitive to sCD4, while non-macrophage-tropic Envs were significantly more resistant. In contrast, all Envs were sensitive to VRC01 regardless of tropism, while MAb b12 conferred an intermediate neutralization pattern where all the macrophage-tropic and about half of the non-macrophage-tropic Envs were sensitive. CD4, b12, and VRC01 share binding specificities on the outer domain of gp120. However, these antibodies differ in their ability to induce conformational changes on the trimeric envelope and in specificity for residues on the V1V2 loop stem and ß20-21 junction that are targets for CD4 in recruiting the bridging sheet. These distinct specificities of CD4, b12, and VRC01 likely explain the observed differences in Env sensitivity to inhibition by these reagents and provide an insight into the envelope mechanisms that control macrophage tropism. We present a model where the efficiency of bridging-sheet recruitment by CD4 is a major determinant of HIV-1 R5 envelope sensitivity to soluble CD4 and macrophage tropism.

A conserved determinant in the V1 loop of HIV-1 modulates the V3 loop to prime low CD4 use and macrophage infection.

The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. Here, we describe a single amino acid determinant in the V1 loop that also modulates macrophage tropism. Thus, we identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, we show that a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection.

Determinants flanking the CD4 binding loop modulate macrophage tropism of human immunodeficiency virus type 1 R5 envelopes.

Human immunodeficiency virus type 1 R5 viruses vary extensively in phenotype. Thus, R5 envelopes (env) in the brain tissue of individuals with neurological complications are frequently highly macrophage-tropic. Macrophage tropism correlates with the capacity of the envelope to exploit low CD4 levels for infection. In addition, the presence of an asparagine at residue 283 within the CD4 binding site has been associated with brain-derived envelopes, increased env-CD4 affinity, and enhanced macrophage tropism. Here, we identify additional envelope determinants of R5 macrophage tropism. We compared highly macrophage-tropic (B33) and non-macrophage-tropic (LN40) envelopes from brain and lymph node specimens of one individual. We first examined the role of residue 283 in macrophage tropism. Introduction of N283 into LN40 (T283N) conferred efficient macrophage infectivity. In contrast, substitution of N283 for the more conserved threonine in B33 had little effect on macrophage infection. Thus, B33 carried determinants for macrophage tropism that were independent of N283. We prepared chimeric B33/LN40 envelopes and used site-directed mutagenesis to identify additional determinants. The determinants of macrophage tropism that were identified included residues on the CD4 binding loop flanks that were proximal to CD4 contact residues and residues in the V3 loop. The same residues affected sensitivity to CD4-immunoglobulin G inhibition, consistent with an altered env-CD4 affinity. We predict that these determinants alter exposure of CD4 contact residues. Moreover, the CD4 binding loop flanks are variable and may contribute to a general mechanism for protecting proximal CD4 contact residues from neutralizing antibodies. Our results have relevance for env-based vaccines that will need to expose critical CD4 contact residues to the immune system.

Variation in HIV-1 R5 macrophage-tropism correlates with sensitivity to reagents that block envelope: CD4 interactions but not with sensitivity to other entry inhibitors.

BACKGROUND: HIV-1 R5 viruses cause most of the AIDS cases worldwide and are preferentially transmitted compared to CXCR4-using viruses. Furthermore, R5 viruses vary extensively in capacity to infect macrophages and highly macrophage-tropic variants are frequently identified in the brains of patients with dementia. Here, we investigated the sensitivity of R5 envelopes to a range of inhibitors and antibodies that block HIV entry. We studied a large panel of R5 envelopes, derived by PCR amplification without culture from brain, lymph node, blood and semen. These R5 envelopes conferred a wide range of macrophage tropism and included highly macrophage-tropic variants from brain and non-macrophage-tropic variants from lymph node. RESULTS: R5 macrophage-tropism correlated with sensitivity to inhibition by reagents that inhibited gp120:CD4 interactions. Thus, increasing macrophage-tropism was associated with increased sensitivity to soluble CD4 and to IgG-CD4 (PRO 542), but with increased resistance to the anti-CD4 monoclonal antibody (mab), Q4120. These observations were highly significant and are consistent with an increased affinity of envelope for CD4 for macrophage-tropic envelopes. No overall correlations were noted between R5 macrophage-tropism and sensitivity to CCR5 antagonists or to gp41 specific reagents. Intriguingly, there was a relationship between increasing macrophage-tropism and increased sensitivity to the CD4 binding site mab, b12, but decreased sensitivity to 2G12, a mab that binds a glycan complex on gp120. CONCLUSION: Variation in R5 macrophage-tropism is caused by envelope variation that predominantly influences sensitivity to reagents that block gp120:CD4 interactions. Such variation has important implications for therapy using viral entry inhibitors and for the design of envelope antigens for vaccines.

Variation of macrophage tropism among HIV-1 R5 envelopes in brain and other tissues.

Human immunodeficiency virus (HIV)-positive individuals frequently suffer from progressive encephelopathy, which is characterized by sensory neuropathy, sensory myelopathy, and dementia. Our group and others have reported the presence of highly macrophage-tropic R5 variants of HIV-1 in brain tissue of patients with neurological complications. These variants are able to exploit low amounts of CD4 and/or CCR5 for infection and potentially confer an expanded tropism for any cell types that express low CD4 and/or CCR5. In contrast to the brain-derived envelopes, we found that envelopes from lymph node tissue, blood, or semen were predominantly non-macrophage-tropic and required high amounts of CD4 for infection. Nevertheless, where tested, the non-macrophage-tropic envelopes conferred efficient replication in primary CD4(+) T-cell cultures. Determinants of R5 macrophage tropism appear to involve changes in the CD4 binding site, although further unknown determinants are also involved. The variation of R5 envelopes also affects their sensitivity to inhibition by ligands and entry inhibitors that target CD4 and CCR5. In summary, HIV-1 R5 viruses vary extensively in macrophage tropism. In the brain, highly macrophage-tropic variants may represent neurotropic or neurovirulent viruses. In addition, variation in R5 macrophage tropism may also have implications (1) for transmission, depending on what role macrophages or cells that express low CD4 and/or CCR5 play in the establishment of infection in a new host, and (2) for pathogenesis and depletion of CD4(+) T cells (i.e., do highly macrophage-tropic variants confer a broader tropism among CD4(+) T-cell populations late in disease and contribute to their depletion?).

Non-macrophage-tropic human immunodeficiency virus type 1 R5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis.

Human immunodeficiency virus type 1 (HIV-1) R5 isolates that predominantly use CCR5 as a coreceptor are frequently described as macrophage tropic. Here, we compare macrophage tropism conferred by HIV-1 R5 envelopes that were derived directly by PCR from patient tissue. This approach avoids potentially selective culture protocols used in virus isolation. Envelopes were amplified (i) from blood and semen of adult patients and (ii) from plasma of pediatric patients. The phenotypes of these envelopes were compared to those conferred by an extended panel of envelopes derived from brain and lymph node that we reported previously. Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of primary macrophages. Highly macrophage-tropic envelopes were predominate in brain but were infrequent in semen, blood, and lymph node samples. We also confirmed that the presence of N283 in the C2 CD4 binding site of gp120 is associated with HIV-1 envelopes from the brain but absent from macrophage-tropic envelopes amplified from blood and semen. Finally, we compared infection of macrophages, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs) conferred by macrophage-tropic and non-macrophage-tropic envelopes in the context of full-length replication competent viral clones. Non-macrophage-tropic envelopes conferred low-level infection of macrophages yet infected CD4(+) T cells and PBMCs as efficiently as highly macrophage-tropic brain envelopes. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node, blood, and semen is striking and contrasts with the current consensus that R5 primary isolates are generally macrophage tropic. The extensive variation in R5 tropism reported here is likely to have an important impact on pathogenesis and on the capacity of HIV-1 to transmit.