Summary The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope-mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.

Human immunodeficiency virus type 1 (HIV-1) enters cells through the chemokine receptors CCR5 (R5 virus) and/or CXCR4 (X4 virus). Loss of N-linked glycans and increased net charge of the third variable loop (V3) of the gp120 envelope glycoprotein have been observed to be important steps towards CXCR4 use. All reported sequences using CCR5 or CXCR4 exclusively, or using both, were gathered from the Los Alamos HIV Database and analysed with regard to the V3 N-linked glycosylation motifs (sequons) and charge. The V3 loop glycan had a sensitivity of 0?98 and a 0?92 positive predictive value in the context of CCR5 use. The difference from X4 was remarkable (P<10 "12). Especially, the sequon motif NNT within the V3 loop was conserved in 99?2 % of the major clades. The results suggest a close association between the V3 loop glycan and CCR5 use and may provide new insight into HIV-1 tropism and help to improve phenotype-prediction models.To enter cells, human immunodeficiency virus type 1 (HIV-1) uses the coreceptors CCR5 and/or CXCR4, together with the T-cell differentiation antigen CD4. Virus using the CCR5 receptor (R5 virus;Berger et al., 1998) or of the non-syncytium-tropic phenotype is suggested to be more transmissible (Zhang et al., 1993), whilst virus using the CXCR4 receptor (X4 virus;Berger et al., 1998) has been associated with disease progression (Connor et al., 1997; Scarlatti et al., 1997).The third variable (V3) loop of the HIV-1 gp120 envelope glycoprotein was recognized early on to play an important role in governing the choice of target cells (Hwang et al., 1991;De Jong et al., 1992;Fouchier et al., 1992;Shioda et al., 1992). Few amino acid substitutions and an increasing net charge of the V3 loop were sufficient to confer a change in cellular tropism in vitro (De Jong et al., 1992;De Wolf et al., 1994). A decreased number of N-linked glycosylation sites (sequons) in gp120, especially within and around the V3 region, has been demonstrated during evolution from the R5 to the X4 phenotype (Pollakis et al., 2001;Polzer et al., 2001Polzer et al., , 2002.Computer-based models have been developed to predict the biological phenotype of HIV sequences (Briggs et al., 2000;Resch et al., 2001;Jensen et al., 2003;Pillai et al., 2003), based on multiple linear regression (Briggs et al., 2000), specific amino acids within V3 and overall charge among subtype B viruses (Resch et al., 2001) and other subtypes (Pillai et al., 2003). Machine learning has been used to develop phenotype classifiers (Resch et al., 2001) and position-specific scoring matrices have also been used (Jensen et al., 2003).These methods relate to the assumption or possibility that the R5 and X4 phenotypes may be evaluated or classified, based on the properties of their specific amino acids, from the same scale or sum of scales. If the R5-and X4-specific properties relate to different characteristics, however, such programs may overlook essential differences in the R5 versus X4 phenotypes.In a previous study (Clevestig et al., 2005), a phylogenet...

Plasma cells (PCs) are defined as terminally differentiated B cells that secrete large amounts of immunoglobulin (Ig). PCs that reside in the bone marrow (BM) are responsible for maintaining long-term antibody (Ab) responses after infection and vaccination, while PCs present in the blood are generally short-lived. In rhesus macaques, a species frequently used for the evaluation of human vaccines, B cells resemble those found in humans. However, a detailed characterization of BM-resident rhesus PC phenotype and function is lacking. Here, we examined Ig secretion of distinct rhesus CD138+ populations by B cell ELISpot analysis to couple phenotype with function. We demonstrate that the CD20low/−CD138+CD31+ BM population was highly enriched for antibody-secreting cells with IgG being the predominant isotype (60%), followed by IgA (33%) and IgM (7%). Transmission electron microscopy analysis confirmed PC enrichment in the CD20low/−CD138+CD31+ population with cells containing nuclei with “spokes of a wheel” chromatin structure and prominent rough endoplasmic reticulum. This panel also stained human BM PCs and allowed a clear distinction between BM PCs and short-lived peripheral PCs, providing an improved strategy to isolate PCs from rhesus BM for further analysis.

Human immunodeficiency virus type 1 has several genetic subtypes and two coreceptor use phenotypes: R5 that uses CCR5, while X4 uses CXCR4. A high amino acid charge of the envelope glycoprotein 120 V3 region, common at positions 11 and 25, is important for CXCR4 use. We characterized charged V3 amino acids, retrieving all biologically phenotyped sequences from the HIV Sequence Database. Selecting individually unique ones randomly yielded 48 subtype A, 231 B, 180 C, 37 D and 32 CRF01_AE sequences; 482 were R5 and 46 were X4. Charged amino acids were conserved in both R5 and X4 with general and subtype-specific patterns. X4 viruses gained a higher charge from positive amino acids at positions other than in R5, and through the loss of negative amino acids. Other positions than 11/25 had a greater impact on charge (P,0.001). This describes how R5 evolves into X4 in a subtype-specific context, useful for computer-based predictions and vaccine design.To determine the biological function by the amino acid sequences, generates a need to correctly couple these sequences to the appropriate function. For the highly variable human immunodeficiency virus type 1 (HIV-1), such conformity is crucial.Despite the genetic diversity of HIV-1 in subtypes, subsubtypes and recombinant forms, there are only two major phenotypes for cell entry: R5 and X4 (and dual-tropic R5X4), depending on if the virus uses the chemokine coreceptors CCR5, CXCR4 or both (Berger et al., 1998). Previously, such HIV-1 phenotypes were related to whether the cells were syncytium-inducing (SI) or non-syncytiuminducing (NSI), or expressed macrophage (M-tropic) or Tcell (T-tropic) tropism. These three overlap without being interchangeable (Berger et al., 1998).The NSI phenotype appears early in infection and is related to transmission (Zhu et al., 1993), while the SI is associated with disease progression (Koot et al., 1993). The same has been shown for the R5 and X4 phenotypes (Connor et al., 1997; Scarlatti et al., 1997;Casper et al., 2002).HIV-1 subtypes differ epidemiologically and clinically. Subtype C is the most common. Subtype B has dominated among HIV-1-positive intravenous drug users and men who have sex with men. Subtype D is primarily found in central Africa, where subtype A is also prevalent. Subtype D had a larger proportion of CXCR4-using viruses than subtype A (Huang et al., 2007), and was associated with a faster disease progression than A, B or C (Vasan et al., 2006;Baeten et al., 2007;Kiwanuka et al., 2010; Easterbrook et al., 2010). Circulating recombinant form (CRF) 01_AE is spreading in South-east Asia.The HIV-1 envelope glycoprotein 120 (gp120) third variable region (V3) is important for viral tropism (Hwang et al., 1991). Substitutions of single amino acids within V3 affect the NSI/SI phenotype in subtypes A, B, C, D and CRF01_AE (de Jong et al., 1992b;Fouchier et al., 1992;De Wolf et al., 1994). Positively charged V3 amino acids have been associated with the SI phenotype (de Jong et al., 1992a) and M-tropic viruses with a lower V3 net...