*3.4. The BCG.HTI2auxo.int prime-ChAdOx1.HTI Boost Regimen Elicits HIV-1-Specific T-cell Responses*

In order to assess the enhancement of cellular immune responses provided by a prime vaccination with BCG.HTI2auxo.int, adult mice (seven-week-old, *n* = 8/group) were immunized with either 10<sup>5</sup> cfu of BCG.HTI2auxo.int (id) and boosted with ChAdOx1.HTI 10<sup>9</sup> viral particles (vp) delivered intramuscularly (im) after five weeks (group A), or with 10<sup>6</sup> BCGwt (id) and boosted with ChAdOx1.HTI (109 vp, im) after five weeks (group B), or only immunized with ChAdOx1.HTI (10<sup>9</sup> vp, im) at week five (group C), or left unimmunized (group D). The groups and immunization regimens are illustrated in Figure 4A. A group primed with BCGwt was included to allow comparison of the unspecific adjuvanticity of BCG and the specific priming of BCG expressing HTI. Two weeks post-boost, mice were sacrificed and splenocytes were isolated for an ELISpot analysis of IFN-γ secretion in response to 17 peptide pools spanning the HTI proteome. The total magnitude of IFN-γ secreting cells in response to HTI peptide pools was approximately doubled when priming with BCG.HTI2auxo.int as compared to ChAdOx1.HTI alone; however, the same was observed in BCGwt primed mice (Figure 4B). Overall, priming with BCG.HTI2auxo.int or BCGwt increased responses to peptide pools in the responding mice (Figure 4C–E), although these differences only reached trends when compared with animals only receiving ChAdOx1.HTI. In mice immunized with ChAdOx1.HTI alone, statistically significant differences as compared to naïve mice were only observed in response to one pool representing integrase (int) (pool 2C, Figure 4D). Priming with BCG.HTI2auxo.int induced significantly higher responses as compared to naïve mice in response to five HTI-derived pools (Figure 4: 1E p24; *p* = 0.0469, 1H p24; *p* = 0.048, 1K prot; *p* = 0.0004, 2B RT; *p* = 0.0011, and 2C int; *p* = 0.0038). A similar number was observed for mice primed with BCGwt (Figure 4: 1G p24; *p* = 0.0016, 1K prot; *p* = 0.0002, 2B RT; *p* = 0.0004, and 2C int; *p* = 0.0360). However, the IFN-γ response to pool 1E representing p24 was significantly higher in mice receiving BCG.HTI2auxo.int as compared to those receiving BCGwt (Figure 4C). Both the recombinant and wild-type BCG induced *Mtb*-specific responses (PPD, Figure 4E).

**Figure 4.** Induction of HIV-1 specific T-cell responses by the BCG.HTI2auxo.int + chimpanzee adenovirus HIVACAT T-cell immunogen (ChAdOx1.HTI) prime-boost regimen in BALB/c mice. Adult mice (seven weeks old, *n* = 8/group) were immunized with either 105 cfu of BCG.HTI2auxo.int (id) and boosted with ChAdOx1.HTI (10<sup>9</sup> vp, im) after five weeks (group A), or with 10<sup>6</sup> BCG.wt (id) and boosted with ChAdOx1.HTI (10<sup>9</sup> vp, im) after five weeks (group B), or only immunized with ChAdOx1.HTI (109 vp, im) at week five (group C), or left unimmunized (group D). Groups and the immunization schedule are shown in (**A**). Two weeks post-boost, mice were sacrificed, and splenocytes were isolated for enzyme-linked immune absorbent spot (ELISpot) analysis. (**B**) The total magnitude of HIV-1 specific SFCs/10<sup>6</sup> splenocytes was calculated as sums of the SFCs elicited by the 17 HTI peptide pools, the color-coding represents the HIV-1 gene location of the pools. Data are presented as group means and error bars represent the standard deviation of the total sum of SFC/10<sup>6</sup> splenocytes. Statistics were performed using parametric one-way ANOVA. (C-E) HIV-1 and tuberculosis (TB)-specific T-cell responses interferon-γ (IFN-γ spot-forming cells SFC/10<sup>6</sup> in response to HTI-derived peptide pools representing HIV-1 Gag (**C**), HIV-1 Pol (**D**), and Nef, Vif, and tuberculin purified protein derivative (PPD) (**E**). The data are presented as medians of group responses above the threshold. Statistics were performed using the non-parametric Kruskal–Wallis test adjusted for multiple comparisons, \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* <0.001 and \*\*\*\**p* < 0.0001.

Interestingly, a comparison of the average number of reactive HTI peptide pools in vaccinated mice revealed that the number of reactive pools in BCGwt primed mice was significantly lower than in those primed with BCG.HTI2auxo.int (Figure 5A, *p* = 0.0262). This indicates a loss of breadth when priming with BCGwt, even though IFN-γ secreting cells in response to HTI peptide pools were similar when compared with BCG.HTI2auxo.int primed mice. No differences between BCG.HTI2auxo.int primed mice as compared to mice only receiving ChAdOx1.HTI (Figure 5A) were observed regarding the number of pools recognized per mouse, although the mean was slightly increased from 7.3 to 7.8 when priming with BCG.HTI2auxo.int. The highest number of recognized peptide pools was 13 in both groups. Interestingly, certain mice of the BCG.HTI2auxo.int + ChAdOx1.HTI immunized group reacted to pools less frequently recognized by other groups (Figure 5B: 1E p24, 1F p24, 1J prot, 2D int). On the other hand, lower numbers of mice recognized certain peptide pools (Figure 5B: 1G p24, 1H p24, 2E Vif) when compared to mice only immunized with ChAdOx1.HTI.

**Figure 5.** Differential recognition of peptide pools in BCG.HTI2auxo.int + ChAdOx1.HTI immunized BALB/c mice. Adult mice (seven weeks old, *n* = 8/group) were immunized with either 10<sup>5</sup> cfu of BCG.HTI2auxo.int (id) and boosted with ChAdOx1.HTI (109 vp, im) after five weeks (group A), or with 10<sup>6</sup> BCG.wt (id) and boosted with ChAdOx1.HTI (10<sup>9</sup> vp, im) after five weeks (group B), or only immunized with ChAdOx1.HTI (109 vp, im) at week five (group C), or left unimmunized (group D). Two weeks post-boost, mice were sacrificed, splenocytes were isolated for ELISPOT analysis, and the numbers of reactive peptide pools (total n peptide pools =17) were compared for each mouse. (**A**) The number of reactive pools per mouse. (**B**) The number of reactive mice (eight mice per group) in each group according to peptide pool and HIV-1 gene location. Statistics were performed using parametric one-way ANOVA, \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, \*\*\*\**p* < 0.0001.
