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

HIV-1 vaccines need to induce broadly neutralizing antibodies (bnAb) against conserved epitopes in the envelope glycoprotein (Env) to protect against diverse HIV-1 clades. To achieve this, we need to understand how different amino acids affect the Env trimer structure to find a common strategy to readily produce Env vaccines of different subtypes. Previously, using a saturation mutagenesis strategy we identified single Env substitutions that open the CD4bs without modifying the trimer apex. One of these substitutions was a tryptophan residue introduced at position 375. Here, we introduced 375W into a large panel of 27 T/F, acute stage, chronic infection, and AIDS M-tropic, and non-M-tropic primary isolates from clades A, B, C, D and G, and circulating recombinant forms (CRFs) (CRF02_AG, and CRF01_AE), and a complex (cpx) (CRF13_cpx). To understand the effect of 375W mutation on Env trimer structure and tropism, we evaluated soluble (sCD4) and monoclonal antibody (mAb) neutralization of wt and mutant Env+ pseudovirions using bnAbs (b6, 17b, b12, VCR01, 3BNC117, PGT128, 10-1074, PGT145, PG9 and PG16), as well as macrophage infection. Broadly neutralizing Abs (bnAbs) such VCR01, and 3BNC117 neutralized almost all the primary isolates tested while the other bnAbs neutralized many but not all of our panel. In general, 375W did not impair or abrogate neutralization of potent bnAbs. However, b12 and VCR01 showed some tendencies to neutralize 375W macrophage-tropic (mac-tropic) and intermediate mac-tropic mutants more efficiently compared with non-mac-tropic mutants. We identify wt and 375W mutant Envs in our panel that infected macrophages more efficiently than non-mac-tropic variants but did not reach the levels of highly macrophage-tropic brain reference Envs. These partial mac-tropic Envs were classified as intermediate mac-tropic variants. Surprisingly, we observed a mac-tropic (clade G) and intermediate mac-tropic (clade C, and D) primary isolates wt Envs that were not derived from the central nervous system (CNS). The 375W substitution increased sensitivity to sCD4 in all Envs of our panel and increased macrophage infection in many Envs tested including a CRF01_AE X4 variant. However, variants already highly mac-tropic were compromised indicating the presence of other factors implicated in mac-tropism. Increased sCD4 sensitivity and enhanced macrophage infection provide strong evidence that 375W confers exposure of the CD4bs across Envs from different clades/CRF/cpx and disease stages. Enhanced exposure of the CD4bs by 375W had little or no effect on exposure and sensitivity of CD4bs epitopes targeted by potent bnAbs. In summary, we show that 375W consistently increases Env binding to CD4 for diverse Envs from different clades and disease stages, 375W exposure of CD4 receptor is a biologically functional substitution that alone confers mac-tropism on non-mac-tropic Envs and 3) 375W is an ideal substitution for inclusion into HIV vaccines constructed from different subtype Envs, with the aim to elicit neutralizing antibodies that target the CD4bs while maintaining exposure of other Env broad neutralization sites, and 4) we found mac-tropic and intermediate mac-tropic Envs from blood indicating that these Envs could evolve outside of CNS or be released from Brain. Significance Substitutions 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 in development of multi-clade Envelope vaccines. We show 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 central nervous system (CNS). Here, we show that intermediate (clade C, and D) and macrophage-tropic (clade G) Envelopes can be detected outside CNS. Vaccines targeting the CD4bs will be particularly effective against such strains and CNS disease.