The C3/465 glycan hole cluster in BG505 HIV-1 envelope is the major neutralizing target involved in preventing mucosal SHIV infection.

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.

Intradermal but not intramuscular modified vaccinia Ankara immunizations protect against intravaginal tier2 simian-human immunodeficiency virus challenges in female macaques.

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

T cell-inducing vaccine durably prevents mucosal SHIV infection even with lower neutralizing antibody titers.

Recent efforts toward an HIV vaccine focus on inducing broadly neutralizing antibodies, but eliciting both neutralizing antibodies (nAbs) and cellular responses may be superior. Here, we immunized macaques with an HIV envelope trimer, either alone to induce nAbs, or together with a heterologous viral vector regimen to elicit nAbs and cellular immunity, including CD8+ tissue-resident memory T cells. After ten vaginal challenges with autologous virus, protection was observed in both vaccine groups at 53.3% and 66.7%, respectively. A nAb titer >300 was generally associated with protection but in the heterologous viral vector + nAb group, titers <300 were sufficient. In this group, protection was durable as the animals resisted six more challenges 5 months later. Antigen stimulation of T cells in ex vivo vaginal tissue cultures triggered antiviral responses in myeloid and CD4+ T cells. We propose that cellular immune responses reduce the threshold of nAbs required to confer superior and durable protection.

Enriched rat primary ventral mesencephalic neurons as an in-vitro culture model.

Primary ventral mesencephalic (VM) neuronal cultures are gaining importance in the study of molecular mechanisms leading to Parkinson's disease and development of treatment strategies. In practice, these neurons are cocultured with glial cells, making assessment of neuronal specific proteomic and genomic analyses difficult. Hence, development of VM neuron-enriched culture is indispensable for such analyses. In the current study, VM neurons with less than 5% of glial cells in culture were found to survive for 9 days in vitro (DIV), followed by a sudden death phase resulting in less than 5% of neuronal viability. Analysis of expression of precursor and mature neuronal markers, Nestin and MAP-2, respectively, has shown that these VM neurons attain maturity at the 7th DIV both in the presence and in the absence of glial cells. This VM neuron-enriched culture was shown to be rich in dopaminergic neurons from 7th DIV and survived up to 10th DIV. Reactive oxygen species and DNA damage estimated using CMH2CDFDA dye and comet assay, respectively, showed an increase in their respective levels at 9th DIV. Furthermore, expression of topoisomerase II ß, a key player in neuronal development, was found to increase until 9th DIV, followed by a sudden decrease on 10th DIV. In conclusion, the above results provide a good working model of VM neurons in vitro along with 7th DIV as an ideal time period to study and evaluate the pro/antisurvival effects of various compounds on VM neurons.