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.


Mycoplasma contamination
The cell lines were periodically examined for mycoplasma contamination using ATCC's cell authentication service and were negative.

Commonly misidentified lines (See ICLAC register)
No commonly misidentified cell line was used in the study.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
All animals used in the study were female Rhesus Macaques (Macaca mulatta) of India origin, aged 3 -15. A descriptive table containing age and MHC-I alleles are provided in supplementary table 1.

Wild animals
Study did not involve wild animals.

Field-collected samples
Study did not involve samples collected from the field.

Ethics oversight
Animals were maintained as per NIH guidelines and all procedures were approved by the Institutional Animal Care and Use Committee of Emory University. Reported in the first paragraph of the methods section.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

October 2018
Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation PBMCs were prepared from blood collected in BD cell preparation tubes using a standard protocol optimized in the lab. Briefly, the blood samples were centrifuged at 2,000 g for 20 min at room temperature. Plasma was removed and the cells were resuspended in PBS. The cells were transferred to a 15 or 50 ml centrifuge tube, washed once with PBS. The red blood cells were lysed using ACK lysis buffer. The cells were washed thrice and resuspended in complete media for use. For staining, 1 -2 million cells were washed once with PBS and stained with a viability dye followed by staining with a surface antibody cocktail in 100 ul for 30 min at room temperature. The cells were then washed, fixed and permeabilized with cytofix/ cytoperm buffer for 10 minutes. The permeabilized cells were stained with ICS antibodies in perm/wash buffer for 30 min at room temperature. Cells were then washed twice with perm/wash buffer and once with staining buffer before acquisition. The vaginal tissues harvested from animals undergoing necropsy were shipped overnight in cold RPMI supplemented with 10% FBS and antibiotics. The vaginal tissue slices collected after stimulation with DMSO or the Gag peptide pool were minced and digested in 1 mg/ml collagenase type IV. They were then dissociated using a gentleMACS dissociator (Milteyni Biotec) with the settings to dissociate mouse spleen. The dissociation protocol was run three times, and the samples were filtered through 100 μm strainers. The cells were washed twice with Hank's balanced salt solution (HBSS) with 2% FBS. The single cell suspensions were stained with fixable viability dye in PBS followed by staining with a cocktail of oligo-labelled antibodies (shown in Supplementary Table 2) and FITC anti-CD45 (clone D058 -1283) and PE-CF594 anti-CD3 antibodies.

Instrument
BD LSRII for analysis. BD Aria III for live-dead sorting Software BD FACSDiva V8.0 Cell population abundance The primary objective of the sorting was to obtain viable cells for single-cell RNA sequencing. We used this step to enrich for non-T cells. Purity was not of relevance as we mixed 3000 -6000 viable cells for CITE-seq single cell seuqencing.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.