The study protocol was approved by the Scientific Advisory Committee of the ICMR-National Institute for Research in Tuberculosis (NIRT), Chennai, India, and the study was conducted in accordance with Good Clinical Laboratory Practice (GCLP) guidelines. The study protocol was also reviewed and approved by the Institutional Ethics Committee of ICMR-NIRT (TRC IEC No: 2009009) and the Institutional Review Board of the Y. R. Gaitonde Centre for AIDS Research and Education (YRG IRB No: 279), Chennai, India.

The study was carried out on samples collected during the period 2016-2018. Sample collection was performed at YRG CARE, one of the largest tertiary referral HIV Care Centres in southern India, providing medical care and support for more than 22,000 patients, and the study was conducted at ICMR-NIRT. The study population comprised of 4 groups of individuals: (i) HIV-2 infected persons (HIV-2; n=37), (ii) HIV-1 infected individuals on antiretroviral therapy (HIV-1+ART+; n=10),(iii) HIV-infected individuals naïve to ART (HIV-1+ART-; n=10), and (iv) HIV--unexposed uninfected healthy women (HU; n=35). Individuals in groups 1 & 2 received ZDV (Zidovudine) + 3TC (Lamivudine) + NVP (Nevirapine) or TDF (Tenofovir) + 3TC (Lamivudine) + EFV (Efavirenz) as per the National Technical Guidelines on Antiretroviral Treatment (NACO, 2018). In addition, we also included a group of HIV exposed seronegative (HESN) individuals who were HIV-uninfected female spouses of HIV-1 seropositive men (HIV discordant couples; n=35) from one of our earlier studies (unpublished data) for comparison. Enrolment into the study required the willingness of participants to provide written informed consent for specimen collection and storage. All HIV-2 infected persons were free of HIV-1 co-infection at the time of sampling ( Table 1 ). Diagnosis and confirmation of HIV-2 infection was based on serological and molecular diagnostic tests. Serological testing was done using the Retro quick rapid test (Qualpro Diagnostics) and HIV-1/2 Tridot (J. Mitra). Samples were further tested using molecular tests to confirm the absence of HIV-1 co-infection with negative detection in HIV-1 DNA-PCR and positive detection in HIV-2 gene-specific PCR.

Ten milliliters of blood was collected from all study participants by venepuncture in a green top VACUTAINER® containing sodium heparin as the anticoagulant and used for the isolation of peripheral blood mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation and cryopreserved at <-190°C.

Cryopreserved PBMC obtained from the study participants were used for immunophenotyping by flow cytometry. Briefly, two million cells were washed with FACS buffer and stained with Live/Dead Fixable Aqua blue dye (Invitrogen). The following cocktail of monoclonal antibodies were used to enumerate the different cell types: T follicular helper and Treg panel: CD3-APC H7 (SK7), CD4-BUV 737 (SK3), CD8-APC R 700 (RPA-T8), CD45RO BUV395 (UCHL-1),CCR7-PEcy7 (G043H7), CXCR3-PERCP Cy5.5 (IC6), CXCR5-BB515 (RF8B2), PD-1-PE (EH12.1), CD25-APC (A251) and CD127-PECF594 (HIL.7R.M21); B cell panel: CD3-APC H7(SK7), CD38-APC (HIT2), CD20-PE (HIB19), CD19-BUV395 (3H7), IgD-BUV737 (IA6.2), CD27-BB515 (0323; Memory T cell and T_SCM panel: CD3-APCH7 (SK7), CD8-APCR700 (RPA.T8), CD4-BUV737 (RPA-T4), CD45RO-BUV395 (UCHL-1), CCR7-PEcy7 (G043H7),CD95-PECF594 (DX2), CD28-APC (28.2), CD122-PE (MIKB2). NK cell panel: CD3-APCH7 (SK7), CD56-APC(B519), CD16-BUV737 (3CG8), CD95- PECF594 (DX2), NKG2D-PERCP CY5.5 (1D11). Cells were stained with the antibodies for 20 minutes at 4°C (antibody and clone description are provided in Table S1 ). About 2 × 10⁶ cells were stained for each panel. After staining, the cells were washed, fixed with BD Cytofix (2% paraformaldehyde), and analyzed on a FACS ARIA III SORP flow cytometer (Becton Dickinson). A minimum of 1,000,000 total events were acquired, and data were analyzed using FlowJo software, version 10.5 (Tree Star Inc., Ashland, Oregon, USA).

Statistical analyses were performed using GraphPad Prism, version 7.05 (GraphPad Software, Inc., CA). Values are presented as median and interquartile range. Percentage frequency of the immune cell subsets like memory T cells, follicular helper-like CD8⁺ T cells (CXCR5+ CD8⁺ T cells), T_SCM cells, B cells and NK cells were compared between the HIV-2, HIV-1, HESN and HU groups using Kruskal-Wallis test, followed by subgroup analysis using Dunn’s multiple comparison test. Correlation analysis was performed to determine the relationship between the frequency of different immune cell types and broadly neutralizing antibodies titer. For all analyses, differences were considered significant if the p-value was <0.05.

Recent studies suggest that follicular CXCR5 ⁺ CD8⁺ T cells and CD8⁺ stem-like memory cells are involved in the control of HIV/SIV infection (29, 30). We therefore, evaluated the frequencies of these T cell subsets in HIV-2 infected individuals and compared it with that seen in HIV-exposed seronegative (HESN) individuals, HIV-1 infected persons on antiretroviral treatment (HIV-1+ART+) as well as those not on treatment (HIV-1+ART-), and HIV-unexposed healthy individuals (HU). Polychromatic flow cytometry was used to enumerate the different T cell subsets, including circulating Tfh cells, CXCR5 expressing CD8⁺ T cells, stem-like memory T cells, and regulatory T cells. Tfh and CXCR5 expressing CD8⁺ T cells were defined using CXCR5 and PD-1, T stem-like memory cells were identified using CD28 and CD95 expression on naïve T cells, and T regs were identified based on the expression of CD25 on CD127^- CD4⁺ T cells ( Figure 1A ).

We found significantly elevated numbers of CD4⁺ Tfh cells in HIV-2 infected individuals (median: 1.21%; range: 0.70-1.56%) as compared to HIV-1 infected persons (median: 0.58%; range: 0.04-1.04%; p<0.05) as well as unexposed healthy individuals (median: 0.37%; range: 0.23-0.44%; p<0.001) ( Figure 1B ). Incidentally, we also found higher numbers of CD4⁺ Tfh cells in the ESN group (median: 0.86%; range: 0.61-1.27%) as compared to the HIV-infected (median: 0.58%; range: 0.04-1.04%; p=0.02) and HIV-unexposed healthy control groups (median: 0.37%; range: 0.23-0.44%; p<0.001). Further, we found significantly higher frequencies of CXCR5⁺CD8⁺ T cells in HIV-2 infected individuals (median: 0.90%; range: 0.61-1.10%) as compared to HIV-1 infected individuals on ART (median: 0.32%; range: 0.01-1.01%; p<0.05) as well as HIV-unexposed healthy controls (median: 0.01; range: 0.01-0.04%) (p<0.001) ( Figure 1C ). Collectively, our findings reveal significant expansion of critical T cell subsets that help boost the host immune response leading to better control of the virus and slower course of disease progression in HIV-2 infected individuals.

Earlier studies have reported that the self-renewing and multipotent Tscm cells mature into memory cells or reservoirs of effector T lymphocytes, which continue to persist in the host even in the absence of antigen (31). Hence, expansion of Tscm cells appears to be a very critical component in the control of HIV infection. Very interestingly, we found significantly increased numbers of Tscm cells in both the CD4⁺ and CD8⁺ T cell compartments in HIV-2 infected individuals as compared to HIV-1-infected persons ( Figures 1D, E ). The median frequency of circulating CD4⁺ Tscm cells in the HIV-2 group was 1.80% (range: 1.10-2.10%), HIV-1 infected group on ART was 0.8% (range: 0.31-1.80%), HIV infected group naïve to ART was 1.20% (range: 0.70-2.10%), and unexposed healthy groups was 0.63% (range: 0.01-1.45%) respectively. The median frequency of circulating CD8⁺ Tscm cells in the HIV-2 group was 1.70% (range: 1.21-2.21%), HIV-1 infected group on ART was 0.877% (range: 0.26-1.23%), HIV infected group naïve to ART was 0.82% (range: 0.21-1.81%), and unexposed healthy groups was 0.56% (range: 0.23-1.17%). Another striking observation was the presence of a significantly higher frequency of CD8⁺ Tscm cells in the HESN population (median 1.26%; range: 0.71-1.93%) as compared to the HIV-1 infected and HU groups.

In contrast, we observed a decreased frequency of Treg cells in the HIV-2 and HESN groups as compared to the other groups. The ART-naive HIV-infected group had a significantly higher proportion of CD4⁺ Treg cells as compared to the other study groups (p<0.01) ( Figure 1F ). We further analyzed the correlation between the Tregs and memory T cell subsets and found an inverse correlation between Tregs and effector memory T cells in both CD4⁺ (r-0.4, p=0.04) and CD8⁺ (r-0.4, p=0.05) T cell compartments in HIV-2 infected individuals ( Figures 1G, H ). These findings clearly indicate that increased frequency of Tregs help maintain immune homeostasis by controlling the exaggerated immune response and promoting the development of long-lived memory cells during HIV-2 infection.

We analyzed the frequency of different memory B cell subsets in the study groups. Surface markers CD27 and CD38 were used to identify plasmablast B cells (CD19+CD38+CD27+). Memory B cell subsets were distinguished based on IgD and CD27 expression as unswitched memory B cells (IgD+CD27+), class switched memory B cells (IgD- CD27+), atypical memory B cells (IgD-CD27-), and naive B cells (IgD+CD27-) as described by Kaminski et al. (32) ( Figure 2A ). We found a higher frequency of short-lived antibody-secreting plasmablast cells in the HIV-2 positive (median:1.91%; range 1.50 - 2.40%) as well as HESN (median:1.44%; range: 1.05 - 2.14%) groups as compared to treated HIV-1 infected and healthy uninfected groups (median: 1.01%; range: 0.64 -1.69%; p<0.001). Numbers of plasmablast cells were also significantly higher in ART-naïve HIV-1-infected individuals as compared to treated individuals (median:1.95%; range: 1.30- 3.61% p<0.05) ( Figure 2B ). These observations suggest that HIV infection, in general, is associated with an increase in the numbers of antibody-secreting plasmablast cells that further differentiate into long-lived plasma cells and contribute to better control of infection, but their role in slowing down disease progression remains to be clarified.

The HIV-2 and HESN groups were found to possess significantly higher frequencies of class-switched memory B cells and IgM memory B cells (unswitched) as compared to the HIV-1 and HU groups (p<0.001). The median frequency of class-switched memory B cells was 18.2% (range: 12.5 - 23.2%) and unswitched memory B cells was 27.6% (range: 22.8 - 35.4%) in the HIV-2 group ( Figures 2C, D ). We observed a significantly higher proportion of atypical memory B cells in both HIV-1+ART+ and HIV-2 groups as compared to the other groups ( Figure 2E ).

We found a direct correlation between the frequency of atypical memory B cells and the titer of neutralizing antibodies in HIV-2 infected persons (r=0.61, p=0.002) ( Figure 2F ) (neutralizing antibody titres of HIV-2 infected persons is provided in Supplementary Table 1). Levels of atypical memory B cells also correlated positively with the frequency of class-switched memory B cells and central memory CD4⁺ T cells (r=0.48, p<0.019 and r=0.77, p<0.001 respectively) ( Figures 2G, H ). These observations provide further evidence to suggest that early HIV-2 infection is associated with increased CD4⁺ T cell help for B cell differentiation resulting in the development of increased numbers of memory B cell subsets as well as antibody-secreting plasma B cells, thus constituting a robust immune response to fight the infection.

We examined the distribution of memory CD4⁺ and CD8⁺ T cells in HIV-2 infected individuals, given the potential role of these cell types in slowing down disease progression. Effector memory cells are characterized by the expression of C-C chemokine receptor type-7 and CD45RO on their cell surface. Based on their function, 4 distinct populations of effector cells have been described by Gattinoni et al. (31). These include the central memory (CM) T cells defined as CD3+CD4+/CD8+CD45RO+CCR7+ cells, effector memory (EM) T cells defined as CD3+CD4+/CD8+CD45RO+CCR7- cells, terminal effector (TE) T cells defined as CD3+CD4+/CD8+CD45RO-CCR7- cells and naïve T cells (TN) defined as CD3+CD4+/CD8+CD45RO-CCR7+ cells.

The proportion of CD4⁺ and CD8⁺ naïve T cells was significantly lower in the HIV-2, HESN, HIV-1+ART+ and HIV-1+ART- groups as compared to the HU group ( Figure 3A ). On the other hand, the frequency of CD4⁺ effector memory T cells and terminal effector T cells was significantly higher in the HIV-2 and HESN groups ( Figures 3B, C ). While the median frequency of CD4⁺ terminal effector T cells was 18.6 % (range: 12.7- 36.5%) in the HIV-2 group and 14.31% (range: 11.40 - 20.21%) in the HESN group, the median frequency of CD4⁺ effector memory T cells was 43.6% (range: 31.5 - 52.3%) in the HIV-2 group and 44.1% (range: 39.5 - 55.1%) in the HESN group. Earlier studies have also reported a progressive expansion of terminal effector T cells during viral infections, including HIV-1 infection (33–35). We also found that the proportion of cytotoxic function exhibiting terminal effector T cells was very high in HIV-2 infected individuals as compared to HIV-1 infected persons and healthy controls. On the other hand, the proportion of CD4⁺ central memory T cells was higher in the HIV-1 group as compared to the HIV-2 group ( Figure 3D ).

We examined the correlation between the frequency of memory T cell subsets and neutralizing antibody titers in HIV-2 infected individuals. Interestingly, we found a positive correlation between the frequency of CD4⁺ central memory cells as well as effector memory T cells with X731A neutralizing antibodies titer (r=0.54, p=0.007, and r=0.42, p=0.046 respectively) in HIV-2 infected individuals ( Figures 3E, F ). In addition, we noticed a correlation between CD4⁺ central memory T cells and CD4⁺ effector memory T cells (r-0.61, p=0.002) ( Figure 3G ) in the study population.

We also examined the distribution of CD8⁺ memory T cells in HIV-2 infected individuals. As seen with CD4⁺ T cells, naïve CD8⁺ T cells were present at significantly lower levels in the HIV-1 and HIV-2 infected groups as compared to the healthy controls ( Figure 4A ). On the other hand, CD8⁺ terminal effector memory T cells were significantly higher in the HIV-2 and HESN groups as compared to the other groups (p<0.05). The median frequency of CD8⁺ terminal effector T cells was 40.3% (range: 30.4 - 49.4%) in the HIV-2 group and 48.30% (range: 44.31-55.30%) in the HESN group ( Figure 4C ). In contrast, HIV-1-infected persons had a significantly higher proportion of CD8⁺ effector and central memory T cells (p<0.001) ( Figure 4B ). The median frequency of CD8+ effector memory T cells was 44.6% (range: 40.6 - 49.9%) in the HIV-1+ART- group and 38.6% (range: 29.5 - 43.3%) in the HIV-1+ART+ group. The median frequency of CD8⁺ central memory T cells was 24.4% (range: 20.7 - 34.8%) in the HIV-1+ART- group and 32.2% (range: 22.4 - 36.1%) in the HIV-1+ART+ group ( Figure 4D ).

In HIV-2 infected individuals, correlation analysis revealed a significant correlation between the frequency of CD4⁺ effector memory T cells and CD8⁺ central memory T cells (r=0.49, p=0.016) ( Figure 4E ). Similarly, CD4⁺ effector memory T cells correlated significantly with levels of CD8⁺ effector memory T cells and CD8⁺ central memory T cells (r=0.46, p=0.016 and r=0.48, p=0.020) ( Figure 4F ). Levels of CD8⁺ central memory T cells correlated positively with CD8⁺ effector memory T cells (r-0.69, p<0.001) ( Figure 4G ).

NK cell-mediated ADCC is thought to be an essential mechanism involved in controlling HIV infection. Several studies have described NK cell activation and cytolytic function in HIV-1 infected individuals as well as in long-term non-progressors (LTNP) and elite controllers (EC) (36–39). However, the distribution of NK cells and their profile in HIV-2 disease remains mostly undefined. To address this knowledge gap, we characterized the population of NK cell subsets expressing the activation receptor NKG2D and the exhaustion marker CD95 receptor in the study population ( Figure 5A ).

We observed significantly higher frequencies of CD56^negative CD16⁺ NK cells in HIV-2 infected individuals as compared to HIV-1-infected persons and unexposed healthy individuals (p=0.036) ( Figure 5B ). The median frequency of CD56^negative CD16⁺ NK cells in the HIV-2 group was 25% (range:14.1 - 38.5%) and 12.9% (range: 10.4 - 17.1%) in the HIV-1 group. On the other hand, there was no difference in the proportion of CD56^dim CD16⁺ NK cells as well as CD56^bright CD16⁺ NK cells between the groups ( Figures 5C, D ). However, earlier studies have documented a significant reduction in the proportion of mature CD56^dim NK cells as well as immature CD56^bright NK cells in HIV-1 infection (40), with a concomitant increase in the numbers of CD56^negative NK cells (41). Our study demonstrates a greater increase in the proportion of CD56^negative NK cells than CD56^bright and CD56^dim NK cells in HIV-2 infected individuals.

We also found significantly higher numbers of CD56^negative NK cells expressing the NKG2D activating receptor in HIV-2 infected individuals than in HIV-1 and HU individuals (p<0.001 and p<0.05, respectively) ( Figure 5E ). On the other hand, HIV-1 infected individuals had significantly higher numbers of NKG2D expressing CD56^bright and CD56^dim NK cells ( Figures 5F, G ). Similarly, CD95 expressing CD56^bright and CD56^dim NK cells were also significantly more in HIV-2 infected persons as compared to HIV-1-infected individuals and healthy unexposed controls. However, there was no difference between the groups with respect to CD95 expression on CD56^negative NK cells ( Figures 5H-J ).

We also examined the correlation between levels of CD56^negative NK cells and neutralizing antibodies titer as well as other memory T cells and B cell subsets in HIV-2 infected individuals. Interestingly, we found that levels of CD56^negative NK cells correlated significantly with levels of CD27⁺ IgD^- class-switched memory B cells as well as CD27^- IgD^- atypical memory B cells (r-0.70, p=0.0001 and r-0.47, p=0.022) ( Figures 5K, L ). Overall, our results indicate that the unconventional cytotoxic CD56^negative NK cells in HIV-2 infected individuals possess increased activation and reduced exhaustion properties and suggest a possible role for these cells in the slower course of disease progression that is characteristic of HIV-2 infection.