~, response levels were approximately the same between two groups; , one or two of the groups had higher responses. than the mosaic M trimer against clade A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb responses that were comparable to the better component of the mixture for each virus tested. These data suggest that combinations of relatively small numbers of immunologically complementary Env trimers may improve nAb responses. IMPORTANCE The development of an HIV-1 vaccine remains a formidable Rabbit Polyclonal to GFM2 challenge due to multiple circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein vaccine whose sequence has been designed by computational methods to address HIV-1 diversity. The characteristics and immunogenicity of this Env protein, both alone and mixed together with a clade C Env protein vaccine, are described. INTRODUCTION The generation of HIV-1 Env glycoprotein immunogens that can elicit binding and neutralizing antibodies (nAbs) against diverse, circulating HIV-1 strains is a major goal of HIV-1 vaccine development (1,C5). The surface Env glycoprotein, which is the primary target of neutralizing antibodies, comprises the gp120 receptor-binding subunit and the gp41 fusion subunit, and it is present as the trimeric spike (gp120/gp41)3 on the virion surface. During the course of natural HIV-1 infection, nearly all individuals induce anti-Env antibody responses but generally with poor AS-35 neutralization breadth (6,C8). It has been reported that approximately 10 to 25% of HIV-1-infected individuals have the ability to produce broadly neutralizing antibodies (bnAbs) (9). However, a recent evaluation of a large global panel of sera from infected individuals showed that many individuals make nAb responses against a significant fraction of viruses (10). One strategy to address HIV-1 sequence diversity involves the construction of bioinformatically optimized mosaic antigens (11), which are recombined HIV-1 sequences designed for improved AS-35 coverage of global HIV-1 diversity. Several proof-of-concept immunogenicity studies in nonhuman primates have demonstrated that vector-encoded mosaic antigens can augment the depth and breadth of cellular immune responses and also improve antibody responses compared to results with consensus and/or natural sequence antigens (12,C15). We have also recently reported the protective efficacy of vector-based HIV-1 mosaic antigens against acquisition of SHIV-SF162P3 challenges in rhesus monkeys (16). However, the generation of HIV-1 mosaic Env trimers as protein immunogens has not previously been described. In this study, we report the production and characterization of a mosaic M (MosM) gp140 trimer. The mosaic M gp140 trimer bound CD4 as well as multiple bnAbs, including VRC01, 3BNC117, PGT121, PGT126, PGT145, PG9, and PG16, and biophysical studies suggested substantial stability. The mosaic M gp160 also exhibited functional capacity to infect target cells. Immunogenicity studies in guinea pigs showed that the mosaic M gp140 elicited high binding antibody titers, cross-clade tier 1 TZM.bl nAbs, and detectable tier 2 A3R5 nAbs that were a different spectrum than spectra elicited by our clade C gp140 trimer. The nAb response elicited by a mixture of the mosaic M gp140 and our clade C gp140 proved superior to either trimer alone, and the combination induced nAb responses comparable to the better single immunogen in the mixture for each virus tested. MATERIALS AND METHODS Production and expression of mosaic HIV-1 Env proteins. The mosaic M Env gene sequences have been described previously (12, 15, 16). The mosaic gp140s were engineered to contain point mutations to eliminate cleavage and fusion activity (11, 12). To maximize expression in human cell lines, human codon-optimized mosaic M gp140s were synthesized by GeneArt (Life Technologies) with a C-terminal T4 bacteriophage fibritin foldon trimerization domain. A polyhistidine motif was included to facilitate protein purification in one version of the protein. Genes were cloned into AS-35 the SalI-BamHI restriction sites of a pCMV eukaryotic expression vector, inserts were verified by diagnostic restriction digests, DNA was sequenced, and expression testing was performed using 10 g of DNA with Lipofectamine (Life Technologies) in 293T cells. Stable cell lines for natural C clade isolate C97ZA.012 (NatC) (17) and MosM gp140 Env trimers were generated by Codex Biosolutions. For protein production, the stable cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) (supplemented with 10% fetal bovine serum [FBS], penicillin-streptomycin and puromycin) to confluence and then were changed to Freestyle 293 expression medium (Invitrogen) supplemented with the same antibiotics. Cell supernatants.