Increased frequencies of HIV-1-specific T cells after iHIVARNA vaccination
At week 8, patients who had received the highest iHIVARNA dose (group 5) showed a moderate increase in T-cell responses spanning HTI sequence (IN) at week 8 whereas no changes were observed in responses against the rest of the HIV-1 proteome (OUT) compare to baseline (Fig. 3). In addition, the proportion of responders receiving any dose of iHIVARNA (n = 15) increased from 31% (n = 5) at week 0 to 80% (n = 12) postvaccination. This increase was not observed in patients receiving TriMix alone (n = 6) from 50% (n = 3) to 67% (n = 4). The HIV-specific T-cell responses were mainly directed against the following peptide pools: p2 (Gag p17), p4 (Gag p24), p5 (Gag p15), p7 (RT) and p8 (INT), in the IN-peptide pools, whereas in the HIV OUT-peptide pools, the responses were mainly towards: p1 (Gag), p2 (Pol), p4 (Pol), p6 (Env), p7 (Vif, Nef) and p8 (Tat, Vpu, Vpr, Rev; data not shown).
Concerning the analysis of cell subsets, we only observed statistically significant decreases between baseline and week 8 (fourth weeks after the last immunization) in percentages of CD8+ CD38+HLA-DR+ T cells and CD8+ PD-1+ T cells in group 5 (data not shown).
Increased viral expression but stable proviral reservoir after iHIVARNA vaccination
Neither TriMix alone or different doses of iHIVARNA had an impact on proviral HIV-DNA in any of the studied arms (Fig. 4 a). However, there was a transient increase in caHIV-RNA expression at higher doses of iHIVARNA (arms 4 and 5) during weeks 4–6 [whereas this was not observed with TriMix alone (groups 1 and 2) or with low doses of iHIVARNA (group 3)], and subsequently normalized at weeks 8 and 24 (Fig. 4 b). Moreover, the ratio of ca-HIV RNA at week 6 as compared with week-4 levels was significantly higher in patients receiving any dose of iHIVARNA (groups 3–5 merged) vs. patients receiving TriMix alone (P = 0.0126). Finally, usVL also significantly increased at weeks 6 and 8 (P < 0.05) in groups 4 and 5 and returned to baseline values at week 24 (Fig. 4 c). In fact, further analysis of these two groups, showed a positive and significant correlation between the increase of the elicited T-cell immune responses against HTI sequence (IN) and the usVL at week 6 (P < 0.05). No significant correlation was observed between T-cell responses against the rest of the HIV-1 proteome (OUT) and the usVL (See Supplementary Fig. S1, http://links.lww.com/QAD/B364).
iHIVARNA vaccination does not shift gene expression
We did not observe robust differentially expressed genes in any of the group-wise comparisons, although gene set analysis indicates some effect on pathways such as RNA metabolism and host response to viruses, but with very low significance levels (Fig. 5). There were no differentially expressed genes in any of the group-wise comparisons or in the immunologic responders vs. nonresponders, based on ELISPOT assays. Gene set analysis indicated some effect of the vaccine on pathways such as RNA metabolism and host response to viruses. These were related to the presence of a patient effect and an effect of the HIV reservoir size. However, these pathways had very low levels of significance (FDR >0.1), indicating that there was only a modest effect on most pathways.
Recently, direct intranodal administration of naked mRNA has been proposed as an alternative to the immunogens used so far in HIV vaccination trials. As compared with plasmid DNA and viral vectors, mRNA has a better safety profile, can be easily obtained by commercially available kits and stored at room temperature. Additionally, mRNA-mediated gene transfer occurs in nondividing cells and offers the advantage of not being restricted to a subject-specific human leukocyte antigen (HLA) allele . Promising preliminary results have been reported in other infectious diseases and cancer with mRNA vaccines. Indeed, an mRNA influenza vaccine candidate has demonstrated similar efficacy to licensed vaccines in animal models , a prophylactic mRNA-based candidate vaccine against rabies was well tolerated and induced boostable functional antibodies  and direct administration of mRNA has entered clinical testing in cancer [15,16,18,19]. To our knowledge, this is the first in human clinical trial using direct intranodal administration of naked mRNA as a therapeutic vaccine against HIV-1 infection. We have shown that intranodal injection of the iHIVARNA vaccine was feasible, safe and well tolerated. No severe adverse events were observed even with the highest dose of the vaccine, namely group 5 constituting a total of 1200 μg of mRNA (900 μg HTI mRNA and 300 μg TriMix mRNA). Therefore, this dose has been selected for a currently ongoing phase II clinical trial.
The vast majority (98%) of latent viruses in chronic HIV-1-infected patients carry CTL escape mutations that render infected cells insensitive to CTLs directed at standard (canonical) epitopes . It is likely that many of the therapeutic vaccines currently under evaluation expand preexisting clones, which are exhausted and target escape variants. There is specific interest in approaches that stimulate responses against novel, nondominant epitopes [13,29,30]. Mothe et al.  proposed a rational design for the selection of the HIV antigens based on the viral targets of protective HIV-1 specific T-cell responses from three large cohorts of HIV-infected individuals . In addition, two other groups have hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome will induce more efficient immune response than whole protein-based T-cell vaccines [31,32]. Letourneau et al.  designed the HIVconsv immunogen by assembling the 14 most conserved regions of the HIV-1 proteome into one chimeric protein. When delivered in vaccines vectored by MVA and chimpanzee adenovirus, these vaccines were able to shift preexisting immune responses towards conserved, vaccine-encoded regions of HIV in early-treated HIV-infected individuals [29–33]. Our iHIVARNA candidate supports this strategy. The data presented indicate that the vaccine was able to induce moderate HIV-specific immune responses (increase in magnitude and breadth as well as increase in percentage of responders) against overlapping HIV peptide pools, which matched the HTI sequence (’IN’) whereas no augmented responses were observed against peptide pools covering HIV proteins not included in the HTI sequence (’OUT’). However, the Phase I results are not conclusive because of the limited number of patients included in each group.
The ELISPOT assay has been established for the direct ex-vivo quantification of peptide-reactive T lymphocytes from peripheral blood mononuclear cells (PBMC). However, it is true that the predictive power of this assay has been challenged because of the lack of efficacy of some HIV vaccine trials despite the induction of robust Elispot responses . This finding and the emergence of new techniques that have the potential advantage of simultaneously quantifying numerous parameters, raises questions regarding the future role of IFN-γ Elispot in clinical trials of candidate vaccines. Nevertheless, the IFN-γ Elispot assay has been, unlike other techniques, evaluated and validated in several proficiency panels and is advantageous in cost-effectively detecting and mapping T-cell responses . All these benefits are particularly important in a Phase I clinical trial where safety and tolerability were the major end-points.
There is evidence that HIV-1 vaccines are by themselves insufficient to fully harness the stimulatory potential of dendritic cells. It has been suggested that targeting in-vivo dendritic cells by co-stimulatory molecules improves the effectiveness of the vaccines [20,36]. This type of strategy has already been tested in humans with a vaccine co-expressing immune activator molecules. A clinical trial testing a recombinant fowl pox virus vector co-expressing HIV1Gag/Pol and human interferon-γ has been reported [37,38]. In addition, Van Lint et al.  designed mRNAs encoding a mixture of APC activation molecules, referred as TriMix. Dendritic cells modified in vitro or in vivo with TriMix mRNA have been shown to be significantly more immunogenic than unmodified dendritic cells [20,21].
The higher doses of iHIVARNA mRNA might have increased HIV expression as a transient increment in caHIV-RNA expression and usVL were observed. It is likely to be triggered by activation of the immune system through recognition of TLRs. However, the existence of a direct and significant association between the elicited HIV-1 immune response against epitopes included in the vaccine (and not to the rest of the proteome) and the usVL (1 or 2 weeks after the last dose) suggest that it could be secondary to an specific stimulus rather than to an ambiguous and unspecific reaction because of the mere addition of mRNA. Given the limited number of patients, this association needs to be further explored in the ongoing phase IIa clinical trial to be confirmed.
Using whole blood-derived transcriptome analyses, we only observed modest effects on inflammatory pathways. These effects were related to intrinsic differences in the activity of inflammatory pathways between individual patients, rather than to the effect of any of the vaccine formulations. Therefore, the data suggest that any immune activation induced by this vaccine is modest and not detectable by comprehensive transcriptome profiling of whole blood samples.
In conclusion, this phase I exploratory dose-escalating trial showed that iHIVARNA vaccination was feasible, harmless and well tolerated, was able to induce moderate HIV-specific immune responses and transiently increased caHIV-RNA expression and ultrasensitive plasma viremia. These data support further exploration of iHIVARNA in the currently ongoing phase II clinical trial.
This study was partially supported by grants: FP7-HEALTH-2013-INNOVATION-1 Proposal No: 602570–2, SAF2015–66193-R, FIS PI15/00641, FIS PI15/00480, Fondo Europeo para el Desarrollo Regional (FEDER), RIS**.
**RIS: The SPANISH AIDS Research Network RD16/0025/0002- ISCIII – FEDER.
This study was presented in part at the 2018 Conference on Retroviruses and Opportunistic Infections, Boston, USA.
iHIVARNA Consortium: Consorci Institut d’Investigacions Biomédiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain: Lorna Leal, Blanca Paño, Carlos Nicolau, Amparo Tricas, Marta Sala, Encarnación Moreno, Cristina Rovira, Carmen Hurtado, Constanza Lucero, Irene Fernández, Flor Etcheverry, Alberto Crespo, Manel Bargalló, Miriam García, Alexy Inciarte, Ismael Pérez, Laura Mensa, Laura Mendoza, Anna Vanesa Oliveira, Mª Jose Maleno, Agathe León, Maria Joyera, Judit Pich, Jose M Gatell, Joan A Arnaiz, Montserrat Plana, Felipe García.
Instituut voor Tropische Geneeskunde (ITM), Antwerp, Belgium: Guido Vanham, Eric Florence, Jozefien Buyze, Pieter Pannus.
Vrije Universiteit Brussel (VUB), Brussels, Belgium: Kris Thielemans, Joeri Aerts, Sabine Allard, Patrick Lacor, Nik Claesen, Elger Vercayie, Patrick Tjok.
eTheRNA BVBA (eTheRNA), Brussels, Belgium: Carlo Heirman, Sonja Van Meirvenne, Hilde Van Raemdonck, An Van Nuffel, Jacques Berlo, Inge Pettersson, Gust Schols.
Erasmus Universitair Medisch Centrum Rotterdam (EMC), Rotterdam, Netherlands: Rob Gruters, Marion Koopmans, Wesley de Jong, Henk-Jan van den Ham, Patrick Boers, Rachel Scheuer, Eric Van Gorp, Cynthia Lungu, Arno Andeweg, Ab Osterhaus.
irsiCaixa AIDS Research Institute, Badalona, Spain: Christian Brander, Bonaventura Clotet, Marta Marszalek, Sara Moron-López, Beatriz Mothe, Alex Olvera, Miriam Rosas, Maria Salgado, Javier Martinez-Picado, Mireia Manent, Judith Dalmau.
Synapse Research Management Partners S.L. (SYNAPSE), Barcelona, Spain: Carlos Díaz, Montse Camprubí.
Asphalion, S.L. (ASPHALION), Barcelona, Spain: Lídia Cánovas, Núria Coderch, Marta Rayo 9Lunar y Joel Montané
Authors contributions: L.L., J.P., J.A.A., J.M.G. and F.G. conducted the clinical trial. A.C.G. and M.P. conducted the immunogenicity analyses. B.M. and C.B. developed the HTI in the iHIVARNA vaccine. S.M.L., M.S. and J.M.P. were in charge of the reservoir study. C.H., S.V.M. and K.T. developed the Trimix in the iHIVARNA vaccine and quality control for the vaccine. G.V. and P.P. studied the changes in usVL. H.H., R.G. and A.A. were in charge of the transcriptome analyses. All authors contributed in writing and revising the manuscript.
Conflicts of interest
There are no conflicts of interest.
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HIV; HIVACAT immunogen; naked mRNA; therapeutic vaccine; TriMix
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