# Lentiviral vector-based T-cell vaccines against Zika and yellow fever viruses

Pierre Authié, Philippe Souque, Fanny Moncoq, Amandine Noirat, Catherine Blanc, Sylvain Ciret, Maryline Bourgine, David Hardy, Françoise Guinet, Laleh Majlessi, et al.

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Pierre Authié, Philippe Souque, Fanny Moncoq, Amandine Noirat, Catherine Blanc, et al. Lentiviral vector-based T-cell vaccines against Zika and yellow fever viruses. Vaccine, 2025, 62, pp.127563. 10.1016/j.vaccine.2025.127563. pasteur-05235063

HAL Id: pasteur-05235063

https://pasteur.hal.science/pasteur-05235063v1

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# Lentiviral vector-based T-cell vaccines against Zika and yellow fever viruses

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Pierre Authié<sup>a</sup>, Philippe Souque<sup>a</sup>, Fanny Moncoq<sup>a</sup>, Amandine Noirat<sup>a</sup>, Catherine Blanc<sup>a</sup>, Sylvain Ciret<sup>a</sup>, Maryline Bourgine<sup>a</sup>, David Hardy<sup>b</sup>, Françoise Guinet<sup>c</sup>, Laleh Majlessi<sup>a,1</sup>, Pierre Charneau<sup>a,1</sup>, Kirill Nemirov<sup>a,*1</sup>

## ARTICLE INFO

**Keywords:**

Zika

Yellow fever

Flavivirus

Lentiviral vector

Ifnar-/- mice

T-cell vaccine

CD8<sup>+</sup> T cells

## ABSTRACT

Zika and yellow fever flaviviruses induce important human diseases, mainly in tropical regions. Zika virus is an emerging pathogen that has recently spread to new geographic areas, while yellow fever virus, effectively controlled in the past, has recently re-emerged and caused outbreaks in endemic regions. Since antibody-based vaccines could potentially increase the severity of infections by the related Zika and dengue viruses via antibody-dependent enhancement, new vaccine approaches need to be explored. Here, we described lentiviral vectors that express antigens based on T-cell epitope-containing regions of Zika and yellow fever viruses and evaluated the immunogenicity and protective efficacy induced by these vectors in an ifnar-/- mouse model. These vectors induced T-cell responses in the absence of infection-enhancing antibodies and demonstrated significant protection against the two pathogens, associated with reduced viremia and viral load in organs, as well as protection against weight loss and organ pathology.

## 1. Introduction

Zika virus (ZIKV) and yellow fever virus (YFV) are mosquito-transmitted pathogenic flaviviruses present in tropical and subtropical regions that pose a substantial danger for public health worldwide. Between 2007 and 2016 ZIKV has explosively spread to new geographic areas in South America and Southeast Asia causing outbreaks of congenital syndrome in newborns and Guillain-Barré syndrome in adults. Approximately 1.6 million people were estimated to be infected in 70 countries, with some studies suggesting numbers as high as 132.3 million [1,2]. Although the potential for ZIKV re-emergence remains high, no licensed vaccine or effective treatment against this virus is currently available [1,3].

YFV is endemic in 44 countries in (sub)tropical regions of Africa and South America [4]. An estimated 200,000 yellow fever cases per year result in 30,000 deaths and 400–500 millions of unvaccinated people currently live in at-risk geographic areas [5]. Although the majority of human YFV infections are asymptomatic, ≈12 % of yellow fever cases are severe and manifest with jaundice, hemorrhage and multisystem organ failure [6]. The disease was efficiently controlled by the mosquito

control measures and the use of live-attenuated "17D-204" vaccine. However, YFV has gradually re-emerged when the mosquito eradication programs were relaxed [7]. Several large outbreaks of yellow fever that occurred in endemic areas during last years have resulted in shortages of the vaccine supplies. Thus, an easy-to-produce vaccine with improved safety profile is necessary for continuous protection against yellow fever.

ZIKV is closely related to dengue virus (DENV) and has recently spread to DENV-endemic geographic areas, causing outbreaks of the disease with previously unseen severity. This suggests that the severity of Zika could be enhanced by immune responses to previous DENV infections, via the mechanism known as "antibody-dependent enhancement" (ADE) [8]. ADE implies that antibodies generated against surface proteins of a virus that caused primary infection could enhance secondary infection by a related virus [9]. The role of antibody responses induced by primary DENV or ZIKV infections in the enhancement of secondary heterologous infections is well established by experiments in mouse models and epidemiological data [8,10,11]. Although YFV is much more distantly related to DENV and ZIKV, several studies indicated that antibody responses to distantly related flaviviruses could

<footnote><sup>a</sup> Pasteur-TheraVectys Joint Laboratory, Institut Pasteur, Université Paris Cité, Paris F-75015, France</footnote>

<footnote><sup>b</sup> Histopathology platform, Institut Pasteur, Université Paris Cité, Paris F-75015, France</footnote>

<footnote><sup>c</sup> Microenvironment and Immunity Unit, Institut Pasteur, Université Paris Cité, INSERM U1224, Paris F-75015, France</footnote>

<footnote>* Corresponding author at: Pasteur-TheraVectys Joint Laboratory, Institut Pasteur, Université Paris Cité, Paris F-75015, France.
E-mail address: kirill.nemirov@pasteur.fr (K. Nemirov).</footnote>

<footnote n="1">¹ These authors jointly supervised this work</footnote>
either result in ADE [12,13] or inhibit initiation of virus-specific immune responses [14]. Therefore, the risk of disease enhancement due to vaccination against one of these related viruses needs to be considered during vaccine development.

One promising strategy to avoid ADE is to develop vaccines inducing protective T-cell responses, shown to play an important role in protection against flaviviruses [15]. Many protective DENV T-cell epitopes are located in conserved non-structural (NS) proteins that are not targeted by the ADE-inducing antibodies [16]. Although ZIKV-specific T-cell epitopes are located in both structural and NS proteins, prior immunity to DENV was shown to redirect T-cell responses to ZIKV/DENV cross-reactive epitopes, mainly located in NS proteins [17–19]. DENV-specific CD8⁺ T cells targeting such epitopes induced partial protection against ZIKV in mice lacking the receptor for type I interferon (ifnar⁻/⁻), an accepted model of flavivirus infection [20]. Furthermore, ifnar⁻/⁻ mice transgenic for human leukocyte antigens (HLA) immunized with cross-reactive DENV/ZIKV peptides displayed partial protection against ZIKV [21]. Although humoral responses induced by live-attenuated 17D-204 YFV vaccine are considered the main correlate for protection, this vaccine also induces long-lasting polyfunctional T-cell responses that could have protective effect [4,22].

Lentiviral vectors (LVs) are particularly suited for the induction of strong long-lasting T-cell responses, thus providing a promising platform for the development of such “T-cell vaccines” [23]. Recently, we generated a strongly protective LV vaccine expressing a polyantigen based on T-cell epitope-containing regions of DENV NS proteins (LV-DENV) [24]. Here, we report the development, T-cell immunogenicity and pre-clinical proof of concept of LV-based vaccine candidates against ZIKV and YFV, demonstrating strong protective potential against both infections.

## 2. Materials and methods

### 2.1. Antigen design

Phylogenetic trees were constructed with Mega 7 software, and consensus sequences were inferred from the aa sequences using Consensus Maker tool [25]. MHC-I-restricted T-cell epitopes were predicted from the consensus sequences using Proteasomal cleavage/TAP transport/MHC-I binding combined predictor tool at IEDB [26] and netCTLpan predictor [27]. IEDB predictor was used to identify all 9- and 10-mer peptides presentable by 27 most prevalent HLA alleles [28] and candidate epitopes with a total positive score and a cut-off binding affinity (IC50) of ≤500 nM were selected. Predictions with netCTLpan were performed for the same set of HLA alleles for all 9-mer peptides, and 100 epitopes with the best combined prediction score per HLA allele were retained. MHC-I-restricted epitopes predicted by both methods were aligned to the consensus sequences of ZIKV and YFV and plotted along the sequence to identify the regions containing largest number of epitopes. Chosen regions were joined together to create a single peptide that could be expressed as an antigen from a LV. T-cell epitope predictions were performed again on the assembled antigen to verify that epitopes located close to the borders of individual regions would form correctly, and that no non-specific immunodominant epitopes would appear due to linking of the individual regions together. If such epitopes were identified by predictions, they were eliminated by insertion of hydrophobic amino acid linkers at the junction sites.

### 2.2. Production of lentiviral vectors

DNA sequences encoding polyantigens ZIK, YF-NS and YF-S were codon-optimized for expression in mammalian cells and synthesized by GeneCust (France). The synthetic codon-optimized NS1 sequence of Asian ZIKV (strain BeH81905) was described previously [29]. All sequences were inserted into pFLAPΔU3-β2m-WPRE vector between the human β2 microglobulin (β2m) promoter and the mutated Woodchuck

Posttranscriptional Regulatory Element (mWPRE) [30]. LV were produced in Human Embryonic Kidney (HEK)-293 T cells and titrated by qPCR on LV-transduced HEK-293 T cells, treated with aphidicolin to prevent cell division, as described previously [24,31].

### 2.3. Mice

Ifnar1⁻/⁻ mice that carry Ifnar1tm1Agt allele on either 129 (A129) or C57BL/6J (IFNAR-BL6) genetic background, aged 6 to 16 weeks, were obtained from colonies maintained under specific pathogen-free conditions at central animal facilities of Institut Pasteur. All experiments involving ZIKV and YFV infections were performed in a A3 animal facility, in accordance with the European and French guidelines. Experimental procedures were approved by the Institute Pasteur Safety, Animal Care and Use Committee (CETEA no. DAP1800077) and Ministry of High Education and Research (APAFIS#18428-2019010717408411_v2).

### 2.4. Viral strains, their amplification and titration

Asian ZIKV strain H/PF/2013 (GenBank: KJ776791) was obtained through the DENFREE (FP7/2007–2013) consortium. The YFV vaccine strain 17D-204 was obtained from the commercial lot of vaccine (Stamaryl), purchased from the Institut Pasteur vaccination centre. All viral stocks used for infection were produced in Vero E6 cells, grown in T-175 tissue flasks, and titrated on Vero E6 cells as described previously [24].

### 2.5. Immunization and inoculation of mice with ZIKV and YFV

All immunizations of mice with LV-based vaccine candidates were performed by intramuscular (i.m.) injection in 50 µl in the posterior muscle. Inoculation of ZIKV or YFV was performed by intraperitoneal injection (i.p.) of non-anesthetized animals with viral stocks diluted in PBS in a total volume of 300 µl. The inoculation doses of ZIKV and YFV depended on viral titres, and infectivity of the practicable doses for ifnar⁻/⁻ mice was verified in preliminary experiments. The weight of virus-inoculated mice was regularly measured and they were monitored for signs of illness. Mice were considered to have reached humane endpoint either if they lost 20 % of their initial weight, or if the weight loss of 10 % was accompanied by the appearance of neurological symptoms, such as hunched back posture, movement difficulties and flaccid paralysis of limbs. At the end of each experiment all mice were euthanized by cervical dislocation.

### 2.6. Immunoassays

The ELISPOT was performed as described before [24], generally following the protocol supplied with ELISPOT kit for IFNγ detection (Mabtech AB, Sweden), except that 96-well PVDF plates (Millipore, Sigma) were activated by incubation with 35 % ethanol, washed, and coated by overnight incubation with 100 µl per well of 5 µg/ml rat anti-mouse IFNy antibody (clone AN-18, BD Pharmingen).

Analysis of intracellular cytokine secretion (ICS) from mouse splenocytes was performed as previously described [32]. Surface staining was performed with PE-Cy7-anti-CD107a mAb (clone 1D4B, BioLegend), near IR Live/Dead (Invitrogen), PerCP-Cy5.5-anti-CD3e (clone 145-2C11), eF450-anti-CD4 (clone RM4–5, eBioscience), and BV711-anti-CD8 (clone 53–6.7) mAbs (BD Biosciences or eBioscience). CytoKine staining was performed using PE-anti-IL-2 (clone JES6-5H4), FITC-anti-TNF (MP6-XT22) and APC-anti-IFNγ (clone XMG1.2) mAbs (BD Biosciences).

### 2.7. ADE assay

In vitro ADE assay was performed essentially as it was described [33]. Briefly, 100 µl of diluted mouse plasma samples were incubated for
2 h with 100 µl of DMEM, supplemented with 2.5 % FBS and containing either $3 \times 10^7$ PFU of ZIKV or $1 \times 10^6$ PFU of YFV. As a positive control, dilutions of ADE-inducing pan-flavivirus 4G2 mAb, clone D1-4G2-4-15 (Novus biologicals) in PBS were used instead of mouse serum. After the incubation, the mixes were added to human myelogenous leukaemia K-562 cells, plated at $4 \times 10^5$ cells per well in a 96-well plate and incubated at 37 °C, 5 % CO$_2$ for 1 h. After the incubation cells were washed with PBS, resuspended in 200 µl of RPMI containing 2.5 % FBS, and kept at 37 °C for 48 h (for ZIKV) or 96 h (for YFV). At the end of incubation, cells were permeabilized for 20 min with Cytofix/Cytoperm kit (BD Biosciences) and stained during 1 h with FITC-conjugated 4G2 mAb (Novus biologicals). After staining cells were washed, fixed with Cytofix buffer (BD Biosciences) and analysed on an Attune flow cytometer (Invitrogen).

## 2.8. Viremia and viral loads in mouse organs

Blood for the analysis of viremia was collected from immunized and virus-inoculated animals and processed as described previously [24]. Analysis of ZIKV and YFV viremia and organ loads was performed using published two-step qRT-PCR protocols [34,35]. The RT reaction was performed by use of Moloney murine leukaemia virus (M-MLV) reverse transcriptase and the resulting product was used in duplicate qPCR run on a QuantStudio 12 K Flex real-time PCR system (Applied Biosystems, Carlsbad, CA, USA).

## 2.9. Histopathology

Mouse brain and spleen samples were fixed in formalin for 72 h and embedded in paraffin. Five-µm thick paraffin sections were stained with Haematoxylin and Eosin (H&E). Slides were scanned using the AxioScan Z1 (Zeiss) system and images were analysed with the Zen 2.6 software.

## 2.10. Statistical analyses

Significance of the differences was determined by use of the statistical tests implemented in GraphPad Prism 9.5.0 [36]. For pairwise comparisons, unpaired parametric *t*-test was used with or without Welch's corrections, depending on standard deviation (SD) determined from the dataset. Comparative analysis of small sample groups was performed using unpaired non-parametric Mann-Whitney test. Multiple comparisons were performed either using one-way ANOVA test, either ordinary or Welch, depending on sd, or non-parametric Kruskal-Wallis test. Data were considered significant when *p* values were < 0.05.

# 3. Results

## 3.1. Design and refinement of ZIKV and YFV T-cell polyepitopes

To create ZIKV- or YFV-derived polyepitopic antigens, we evaluated genetic diversity of each virus by inferring phylogenetic trees (Fig. 1),

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Principle of ZIKV and YFV antigen design. A phylogenetic tree representing major genetic lineages of ZIKV and YFV. Consensus sequences representing each lineage were inferred based on individual sequences and used to identify regions containing known human MHC-I- and -II-restricted epitopes, as well as predicted MHC-I epitopes. Epitope-containing regions were assembled together and optimized as outlined in the Material and Methods section.</figcaption></figure>
and then generated consensus sequences representing major phylogroups [37,38]. Known T-cell epitopes of ZIKV and YFV, restricted by human Major Histocompatibility (MHC)-I and -II molecules, were retrieved from the immune-epitope database (IEDB) [26] and aligned with consensus sequences. In order to further improve mapping of epitope-containing regions, epitopes presentable by 27 HLA-I alleles shared by 97 % of global human population [28] were also predicted with bioinformatic tools [26,27]. Alignments of known and predicted MHC-I- and -II-restricted epitopes with consensus sequences of ZIKV and YFV allowed us to identify epitope clusters located in evolutionary conserved regions (Fig. 2). Regions with the highest density of such epitopes were assembled, creating ZIKV-specific antigen "ZIK" (Fig. S1) and YFV-specific antigens "YF-S" and "YF-NS" (Fig. S2). To achieve protection against the broadest possible spectrum of ZIKV genotypes with a limited antigen size, we largely based the amino acid (aa) sequence of ZIK on the Asian genotype consensus sequence. This genotype is more prevalent and diverse globally, and it has recently been responsible for several large ZIKV outbreaks. However, within ZIK, we made the E143K and P147A alterations to conform with the African genotype consensus sequence, which encodes more predicted MHC I epitopes, presentable by a larger spectrum of human HLA alleles.

Both YFV-specific antigens were based on the aa sequence of the live-
atenuated vaccine strain 17D-204, since it provides sufficient protec-
tion against all currently circulating YFV strains and shares 97 % amino
acid identity with the consensus sequence of all YFV lineages. That strain
has also been shown to be infectious in *ifnar*−/*−* mice [39] and was used
as a challenge model in our protection experiments. The YF-S antigen
includes T-cell epitopes identified in the structural proteins (C, PrM and
E) and NS1 of YFV, while the YF-NS antigen is comprised by epitopes
located in NS2A, NS3B, NS3, NS4A, NS4B and NS5 proteins. All antigens
were separately cloned into LV expression cassette to generate "LV-ZIK",
"LV-YF-S" and "LV-YF-NS" non-integrative vectors. An additional ZIKV-
specific vector ("LV-ZIK-NS1") was generated to express the secreted
NS1, a well-known factor of flavivirus pathogenesis previously tested as
an immunogen used in multiple vaccine platforms [40].

## 3.2. Immunogenicity of LVs encoding ZIKV or YFV antigens

To evaluate T-cell immunogenicity of the generated LVs, ifnar-/-
A129 mice (n = 5/group) were immunized with a single dose of 1-3 x
10⁸ TU of LV-ZIK, LV-ZIK-NS1, LV-YF-S, LV-YF-NS vaccines or LV-GFP
 irrrelevant antigen). Fourteen days post-immunization splenocytes
from all vaccinated groups, except for LV-GFP, were strongly responsive
by IFNγ production after in vitro stimulation with pools of ZIKV- or YFV-
derived peptides (Fig. 3a-c).

Immunogenicity of LV-ZIK, LV-ZIK-NS1 and LV-YF-NS was also
evaluated in IFNAR-BL6 mice, which are more sensitive than A129 to
flavivirus infections [24,41]. Splenocytes from the vaccinated IFNAR-
BL6 mice were analysed 14 days post-immunization by intracellular
cytokine staining (ICS) of IFNγ/TNF/IL-2 and surface staining of
lymphocyte degranulation marker CD107a after in vitro stimulation
with relevant or negative control peptides (Fig. 3d-g). Antigen-specific,
IFNγ-producing CD8+ T cells were readily detected, as well as IFNγ+
TNF+ or IFNγ+ IL-2+ double positive and IFNγ+ TNF+ IL-2+ triple pos-
itive CD8+ T cells. Notable proportions of CD8+ T cells from vaccinated
mice also co-expressed IFNγ and CD107a. No significant expression of
effector cytokines was observed in mice injected with the control LV.
Much lower levels of IFN/TNF/IL-2 production were also detected in
CD4+ T splenocytes from LV-ZIK-NS1-immunized mice.

3.3. Protective potential of LVs encoding ZIKV- and YFV-based antigens

A129 mice (n = 10) immunized with 1 × 10⁸ TU of LV-ZIK, LV-ZIK-NS1 or control LV were inoculated with 1 × 10⁴ PFU of ZIKV one month later. Only a few visible symptoms of ZIKV infection were noted in these mice, such as ruffled fur and temporary weight loss, exclusively in the

control mice (Fig. 4a). Although viremia was detectable in all animals, it
was significantly lower in the LV-ZIK- or LV-ZIK-NS1-immunized groups
compared to controls at all days post infection (dpi) (Fig. 4b). Viral loads
in spleen and brain, determined at 12 dpi, were at least 100-fold lower in
the vaccinated animals than in the control group (Fig. 4c). Moreover,
infectious virus titres measured in plasma from LV-ZIK- or LV-GFP-
immunized mice at 3 dpi were also significantly different (Fig. 4d).
The absence of infectious virus in any samples from LV-ZIK-immunized
mice suggests that the viral RNA detected in plasma may originate from
non-infectious viral particles.

No weight loss or apparent symptoms were observed in A129 mice
injected with 3 × 10⁸ TU of LV-YF-S, LV-YF-NS or the control LV and
inoculated with 7 × 10⁶ PFU of YFV one month later (n = 10) (Fig. 4e).
YFV inoculation in these mice resulted in low viremia, detectable only in
the control mice (Fig. 4f). Levels of YFV RNA in spleen were also
significantly lower in the mice immunized with YFV-based LV vectors
(Fig. 4g). Moreover, spleens of all control mice demonstrated spleno-
megaly at 5dpi, unlike those of vaccinated animals (Fig. 4h).

Next, IFNAR-BL6 mice (n = 10/group), were injected with 3 × 10⁸ TU of LV-ZIK-NS1 or LV-GFP and inoculated with 1 × 10³ PFU of ZIKV one month later. Total protection of LV-ZIK-NS1-immunized mice against the weight loss, neurological symptoms and death was observed (Fig. S3). In another experiment performed in the same conditions in IFNAR-BL6 mice (n = 5-10/group), LV-ZIK vaccine was also included (Fig. 5a-d). LV-ZIK-immunized mice were totally protected against the weight loss, while small but reversible weight loss was observed in the LV-ZIK-NS1-immunized group (Fig. 5a). No neurological symptoms and no mortality were observed in mice immunized with ZIKV-based LVs, while such symptoms and mortality were detected in 30 % of control mice (Fig. 5b). Significantly lower viremia was detected in mice immunized with LV-ZIK or LV-ZIK-NS1 on 4 and 7 dpi (Fig. 5c). No viral RNA was detectable in the spleen, brain, liver or testes of LV-ZIK-immunized mice on 9 dpi, while it was detectable in the organs of the control mice and the spleens of several LV-ZIK-NS1-immunized mice (Fig. 5d).

Histopathological analysis of the brain and spleen collected on 9 dpi
also revealed marked differences between vaccinated and control groups
(Fig. 6). While the brain tissue of LV-ZIK- and LV-ZIK-NS1-vaccinated
mice appeared largely normal, that of control mice displayed wide-
spread inflammatory lesions consisting of congestive vessels and peri-
vascular infiltrates, sometimes admixed with pyknotic cells, nuclear
fragments and pre-degenerated cells (Fig. 6a,c). In the spleens of all
ZIKV-inoculated mice, the red pulp was occupied by dense cellular
infiltration, showing in some areas signs of cell degeneration and foci of
extramedullary haematopoiesis. Nevertheless, in ZIKV-infected animals
vaccinated with ZIKV-specific vectors, the red pulp infiltration was less
pervasive, leaving a thicker and clearly identifiable ring around the
follicles (Fig. 6b,d,e).

To investigate protective potential of LV-YF-NS, IFNAR-BL6 mice (n = 5/group) were injected with 3 × 10⁸ TU of LV-YF-NS or LV-GFP and inoculated with 5 × 10⁷ PFU of YFV one month later (Fig. 5e-h). Although both groups of mice lost weight during 1-3 dpi, the mean weight of LV-YF-NS-immunized mice increased between 3 and 7 dpi, whereas the weight of the control mice kept decreasing (Fig. 5e). Moreover, 60 % of control mice exhibited signs of severe disease, i.e., weakness and prostration, thus reaching a humane endpoint (Fig. 5f). Viremia was significantly lower in LV-YF-NS-immunized mice at 2 and 4 dpi and undetectable at 7 dpi (Fig. 5g), while still detectable in all control mice. Analysis of viral load in the brain, spleen and liver also demonstrated significantly higher loads in all organs of the control mice (Fig. 5h).

Histological analysis of spleens and brains from control mice inoc-
ulated with YFV revealed cuffing of the blood vessels in the brain
(Fig. 7a) and dense cellular infiltration of the red pulp in the spleen
(Fig. 7b). These pathological changes were also observed in ZIKV-
inoculated control mice. In contrast, no such changes were observed
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Selection of T-cell immunogenic regions of ZIKV and YFV targeted proteins. (a) Scheme of flavivirus polyprotein showing capsid (C), matrix (PrM), envelope (E), NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 proteins. (b and c) Distribution of human T-cell epitopes of ZIKV and YFV, respectively. Each dot shows localization of epitopes within the polyprotein sequences of ZIKV or YFV on X axis, and the number of epitopes that are located at each position of the sequence on Y axis. Top panels: MHC-I- (blue) and MHC-II- (orange) restricted epitopes that are referenced as positive in T-cell assays in IEDB database. Bottom panels: MHC-I-restricted T-cell epitopes predicted from consensus sequences of ZIKV and YFV by IEDB (blue) and netCTLpan (orange) prediction tools. Regions selected for inclusion in each antigen are shaded in yellow (ZIK), green (YF-S) and purple (YF-NS). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_6.png" /></figure>

(caption on next page)
**Fig. 3.** Immunogenicity of LV-ZIK, LV-ZIK-NS1, LV-YF-S and LV-YF-NS vaccine candidates. T-cell responses in A129 mice induced by a single i.m. immunization with (a) $1 \times 10^8$ TU of LV-ZIK, (b) $3 \times 10^8$ TU of LV-ZIK-NS1, (c) $3 \times 10^8$ TU of LV-YF-NS or LV-YF-S, analysed by IFNγ Elispot 14 days post-immunization. Control mice received the same amounts of LV-GFP. Splenocytes of immunized mice were stimulated with pools of synthetic peptides, predicted to be immunogenic in $H-2^b$ mice (Fig. S1 and S2). Splenocytes from LV-ZIK-NS1-immunized mice were stimulated with a single pool of 166 overlapping 15-mer peptides covering the entire NS1 protein from ZIKV (JPT Peptide Technologies GmbH). Statistical significance of the differences between groups was evaluated by either (a, b) Mann-Whitney t-test or (c) one-way Anova test (ns, non-significant, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001). Error bars show group means with standard deviation (SD). (d-g) ICS analyses of T splenocytes expressing IFNγ/TNF/IL-2 and CD107a (n = 2/group) after in vitro stimulation with a negative control or relevant peptides derived from ZIKV, ZIKV-NS1 and YF-NS antigens. (d) Cytometric gating strategy. (e-g) Various effector T subsets in CD8⁺ (e-f) and CD4⁺ (g) splenocytes.

in LV-YF-NS-immunized YFV-inoculated mice.

### 3.4. Absence of ADE-inducing antibodies in mice immunized with LV-ZIK and LV-YF-S

As ZIK and YF-S polyantigens encode regions originating from the structural proteins of their respective viruses, we wondered whether expression of such regions from LVs could induce antibodies capable of enhancing ZIKV or YFV infection. To address this issue, plasma samples of mice immunized with LV-ZIK or LV-YF-S were analysed by in vitro ADE assay (Fig. 8). Diluted plasma samples were pre-incubated with either ZIKV or YFV, then used to infect human K-562 cells [33]. In the absence of mouse plasma, 10 % of ZIKV⁺ and 2.5 % of YFV⁺ cells were respectively detected by cytometry. Pre-incubation of ZIKV with either dilutions of an ADE-inducing monoclonal antibody (mAb), or plasma from non-immunized ZIKV-infected mice, substantially increased the percentages of ZIKV⁺ K-562 cells, indicating an enhancement of ZIKV infection. Similarly, YFV infection was enhanced in the presence of ADE-inducing mAb. Conversely, no increase in the percentages of ZIKV⁺ or YFV⁺ K-562 cells was observed after inoculation with ZIKV and YFV pre-incubated with plasma of mice collected before or 14 days post-immunization with LV-ZIK or LV-YF-S. These results indicate that immunization with LV-ZIK or LV-YF-S did not induce antibodies capable of facilitating ZIKV or YFV infections.

Altogether, our results demonstrate that all tested LVs induced significant protection against symptomatic diseases caused by ZIKV and YFV in *ifnar*−/− mice, reducing the scale and duration of viremia and viral load in organs, as well as protecting mice against weight loss, organ pathology and death.

## 4. Discussion

Many vaccine candidates have demonstrated protection against ZIKV and YFV in preclinical studies. However, most of these candidates are designed to elicit antibody responses against PrM and E surface proteins [4,42]. In contrast, relatively few studies analyze the protective efficiency of vaccines targeting NS proteins [3,40,43,44]. A DNA vaccine encoding the rearranged NS3 protein of ZIKV demonstrated efficacy in protecting pregnant wild-type and *ifnar*−/− female mice and their fetuses from ZIKV-induced mortality and pathological signs of infection. This vaccine also reduced viremia and viral load in organs by $10^{-10^2}$ fold [44]. A subsequent study reported even more significant protection, wherein humanized HLA-B\*0702 *ifnar*−/− mice were immunized by prime-boost injections of an alphavirus replicon-based RNA vaccine encoding the NS3 protein of ZIKV [43].

Mice challenged with either sub-lethal or lethal doses of ZIKV 21 or 42 dpi were protected from death and showed a mean reduction of viremia ranging from $10^5$ to $10^7$ fold. The findings of both studies indicated that NS3-based vaccines did not generate detectable neutralizing or infection-enhancing antibodies. However, they did induce polyfunctional CD8⁺ T-cell responses correlating with protection. Previously, such polyfunctional CD8⁺ T cells secreting IFNγ, TNF, and IL-2 cytokines, and expressing markers of lymphocyte degranulation, granzyme B or CD107a, have been demonstrated to play major protective roles against DENV, ZIKV, and YFV infections in both humans and mouse models [22,45].

Immunization with vaccines expressing the NS1 protein of ZIKV,

either alone or in combination with surface prM and E proteins, has been shown to induce NS1-specific antibody and T-cell responses that were protective against infection in wild-type and *ifnar*−/− mice [40]. Therefore, the immunization of wild-type mice with a modified Vaccinia virus Ankara (MVA) vector expressing the NS1 protein of ZIKV provided protection against a lethal intracranial ZIKV challenge, resulting in a $> 10^5$-fold reduction of viral titers in the brain. Immunization with a DNA vaccine encoding the secretable form of NS1 has been shown to decrease viremia in wild-type BALB/c mice, with a reduction reaching up to $10^4$-fold. However, the effect of this immunization in *ifnar*−/− mice has not been significant in viremia reduction nor in protection against ZIKV-induced death [40]. Finally, the immunization of *ifnar*−/− mice with the recombinant vesicular stomatitis virus (rVSV) expressing NS1 of ZIKV resulted in a $10^{-10^2}$-fold reduction in viral titers in the plasma, brain and spleen. This reduction in viral titers led to protection against a low-dose challenge, but not against a lethal ZIKV challenge [40].

According to the findings of previous studies, vaccine candidates lacking YFV surface proteins have been demonstrated to provide significant, albeit incomplete, protection against YFV. BALB/c mice immunized with vaccinia virus-vectored vaccine that encoded complete NS1, NS2a, and NS2b, as well as parts of E and NS3 proteins of YFV, demonstrated partial protection from mortality (50 %) induced by intracerebral challenge with 17D-204 YFV [46]. Another study has examined protection of BALB/c mice induced by immunization with a recombinant NS1 fragment produced and purified from *E. coli*. That immunization reduced the mortality induced by an intra-cerebral challenge with homologous 17D-204 strain, but did not protect against the challenge with more virulent strains [40].

The present study examined the LV-ZIK, LV-ZIK-NS1, LV-YF-S and LV-YF-NS vaccine candidates in the *ifnar*−/− mice. Our results show that these vaccine candidates induce antigen-specific T-cell responses and significantly protect animals against morbidity associated with ZIKV or YFV challenges.

Specifically, a $10^3-10^4$-fold decrease in viremia was observed in IFNAR-BL6 mice following a single i.m. immunization with either LV-ZIK or LV-ZIK-NS1 and subsequent challenge with ZIKV one month after immunization. No viral RNA was detectable in any organs of mice immunized with LV-ZIK or in the brain of mice immunized with LV-ZIK-NS1. While viral RNA was still detectable in the spleen of some mice immunized with LV-ZIK-NS1, the mean level of viremia was $10^4$-fold lower than in the control group.

In a similar manner, a single i.m. immunization of IFNAR-BL6 mice with LV-YF-NS, followed by challenge with YFV one month later, resulted in the $10^2-10^4$-fold decrease in viremia and $10^2$-fold decrease in viral load in the organs. Furthermore, both immunizations offered protection against ZIKV- and YFV-induced weight loss and mortality, as well as against pathological changes in the brain and spleen, that were exclusively observed in the organs of control mice.

Although the level of protection observed in A129 mice was lower, these mice were protected against the weight loss induced by ZIKV challenge and showed $10^{-10^2}$ fold and $> 10^2$ fold decrease in mean viremia and organ load levels, respectively. A129 mice immunized with LV-YF-S and LV-YF-NS and challenged with YFV exhibited no detectable viremia, had $10^{-10^2}$-fold reduction in viral RNA compared to the control group, and were protected from YFV-induced splenomegaly.

Direct comparison of results from different studies is complicated by the use of varying mouse and viral strains, infectious doses, and
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**Fig. 4.** Protection against ZIKV and YFV in A129 mice. A129 mice (n = 5-7/group) received i.m. either 1 × 10⁸ TU of LV-ZIK, LV-ZIK-NS1 or 3 × 10⁸ TU of LV-YF-S, LV-YF-NS or corresponding doses of LV-GFP. One month later, mice were inoculated i.p. with (a-d) 1 × 10⁴ PFU of ZIKV or (e-h) 7 × 10⁶ PFU of YFV. (a, e) % of initial weight for individual mice presented as means ± SD. (b, f) Viremia expressed as genome equivalents (G.E.)/ml of plasma. (c, g) Viral loads in the organs, expressed as G.E./µg of total RNA. (d) ZIKV viremia at 3 dpi, expressed as PFU/ml. LOD = limit of detection. (h) Spleen weights (mg). Statistical significance of the inter-group differences was evaluated by either Anova or Kruskal-Wallis test (depending on normality of the data) or (d) Mann-Whitney t-test (* p < 0.05, ** p < 0.01, **** p < 0.0001).
ZIKV

a Mean % of initial weight

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b Survival

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c Viremia

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d Viral load (9 dpi)

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YFV

e Mean % of initial weight

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f Survival

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g Viremia

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h Viral load (7 dpi)

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**Fig. 5.** Protection against ZIKV and YFV in IFNAR-BL6 mice. Mice (n = 5–10/group) received i.m. 3 × 10⁸ TU of LV-ZIK, LV-ZIK-NS1, LV-YF-NS or LV-GFP. One month later, mice were inoculated i.p. with (a–d) 1 × 10³ PFU of ZIKV or (e–h) 5 × 10⁷ PFU of YFV. (a, e) % of initial weight, presented as means ± SD. (b, f) Survival. (c, g) Viremia expressed as genome equivalents (G.E.)/ml of plasma and (d, h) viral loads in the organs, expressed as G.E./µg of total RNA. LOD = limit of detection. Statistical significance was evaluated by (a) ordinary Anova test, (b, f) Mantel-Cox test, (c, d) Kruskal-Wallis tests with Dunn's multiple comparisons (black asterisks) and Mann-Whitney test (red asterisks), (e, g, h) Mann-Whitney test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
a

Untreated

ZIKV inoculated

not immunized

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LV-ZIK

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LV-GFP

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b

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c

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d

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e

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(caption on next page)
**Fig. 6.** Histological features of the organs of IFNAR-BL6 mice, immunized with LV-ZIK or LV-ZIK-NS1 and challenged with ZIKV. (a and b) Haematoxylin-eosin (HE) staining of brain (a) and spleen (b) from IFNAR-BL6 mice, detailed in the Fig. 5d, untreated (first column), or inoculated with ZIKV one month post-immunization with LV-ZIK (second column), LV-ZIK-NS1 (third column) or LV-GFP (fourth column). Representative images of 3 mice per group are shown. In a) black arrows indicate vascular cuffing observed in the brain of mice immunized with LV-GFP and inoculated with ZIKV. In b) white arrows indicate location of red and white pulp zones, respectively. (c) Higher magnification of pathological changes observed in brains of mice immunized with LV-GFP and inoculated with ZIKV: (upper left) Intra- and perivascular infiltrates; (upper right) Infiltrate containing many pyknotic cells (black arrows), nuclear fragments (black arrowheads) and pre-degenerative lesions such as perinuclear halos (empty arrow); (lower left) Examples of shrunk condensed nuclei and debris (arrows) in a perivascular infiltrate; (lower right) Degenerative cell lesions in the hippocampus. Parts of the dentate gyrus (DG) and hippocampus (H) are visible on the upper left side and in the centre of the picture. While some degenerative lesions (arrows), can be seen in the former, the latter is almost completely destroyed and mainly made of cell remnants. (d) Degenerative lesions (arrows) and cell death (arrowheads) in the splenic white pulp of mice vaccinated with LV-GFP and infected with ZIKV (left and centre); an extra-medullary hematopoietic focus in the spleen of an ZIKV-infected mouse vaccinated with LV-GFP (right). Numerous large megakaryocytes are visible (arrows). (e) Examples of lighter, activated areas of the spleen (centre, black boxes). Compared to zones of dense, resting-looking lymphocytes (encircled), these areas contain cells with less densely stained nuclei displaying prominent nucleoli. Signs of mitosis can readily be seen (arrowheads) as well as « ghost » cells likely resulting from the selection processes occurring in germinal centres (black arrows). Rounded and paired nuclei containing one small nucleolus each, are suggestive of follicular dendritic cells (empty arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

<figure><img src="image_32.png" /></figure>

**Fig. 7.** Histological features in the organs of IFNAR-BL6 mice immunized with LV-YF-NS and challenged with YFV. HE staining of brain (a) and spleen (b) sections from IFNAR-BL6 mice detailed in the Fig. 5h that were either untreated (left column) or injected with LV-YF-NS (central column) or LV-GFP (right column) and then inoculated with YFV. Representative images of two-to-three animals of each group are shown. White arrows indicate location of red and white pulp zones, respectively. Black arrows indicate vascular cuffing observed in the brain of mice immunized with LV-GFP vector and infected with YFV. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
<figure><img src="image_33.png" /><figcaption>**Fig. 8.** Search for ADE-inducing antibodies in the plasma of LV-ZIK- or LV-YF-S-immunized mice. K-562 cells were preincubation with plasma from A129 mice collected before or after immunization with LV-ZIK or LV-YF-S and were then infected with ZIKV or YFV. (a) Gating strategy. (b) Untreated cells stained with FITC-4G2 mAb (negative control) or inoculated with ZIKV in the absence of plasma. (c) Cells inoculated with ZIKV after pre-incubation with: (i) pre-immune plasma, (ii) plasma of LV-ZIKV immunized mice collected 14 days post-immunization, (iv) an ADE-inducing mAb, or (iv) plasma from ZIKV infected mice collected 22 dpi. (d) Cells inoculated with YFV after pre-incubation with: medium, an ADE-inducing mAb, plasma collected pre- and post-immunization with LV-YF-S or LV-GFP.</figcaption></figure>
immunization schedules. Nevertheless, the levels of protection against ZIKV and YFV observed in the present study are either similar or superior to those reported in previous studies. Although a study on humanized HLA-B*0702 *ifnar*−/− mice reported an even higher level of protection against ZIKV [43], it should be emphasized that ZIK, YF-S, and YF-NS antigens were optimized to elicit a broad CD8⁺ T cell response in humans. Thus, we expect the protection induced by these vectors in humanized *ifnar*−/− mice to be higher than what was observed in the current study.

We did not detect significant engagement of CD4⁺ T cells post-vaccination with any vectors, except for very weak activation of CD4⁺ T cells observed post-vaccination with LV-ZIK-NS1. This finding aligns with our previously published results [24], and most likely results from relatively inefficient antigenic targeting to MHC-II presentation pathway by these vectors. As CD4⁺ T-cell responses have been demonstrated to play a pivotal role in the protection against flavivirus infections in both humans and mouse models [45,47], it may be advantageous to enhance targeting of antigens to MHC-II presentation pathway through the additional modification of lentiviral vectors [48].

In vitro ADE assay results demonstrated that immunization of mice with LV-ZIK or LV-YF-S did not induce antibodies capable of enhancing ZIKV and YFV infections. This finding is notable given that the antigens encoded by these vectors include regions originating from structural proteins of ZIKV and YFV. This finding suggests that the administration of such vaccines is safe and that further improvements could be made by including more T-cell epitope-containing regions from structural proteins. However, despite the extensive utilization of in vitro assays employing human K562 cells to assess the presence of ADE-inducing antibodies in human and mouse sera, this methodological approach has significant limitations. These include the absence of modelling for the involvement of complement and innate immune signaling, as well as IgG subclass bias. Consequently, it will be important to corroborate these findings through alternative methodologies, such as ex vivo ADE tests on mouse bone marrow-derived macrophages and/or in vivo ADE test in a mouse model.

Our study documents relatively short-term protection in mice, measured one month post-vaccination, as this timing is similar to that of other studies in the field. However, earlier research on such LVs has shown that they elicit T-cell responses that persist for a minimum of three months, and are superior in quality to those induced by other viral vectors, e.g. adenovirus type 5 vector [30]. Future pre-clinical testing of lentiviral prophylactic vaccine candidates will evaluate the longevity of the protection provided by T-cell response-inducing lentiviral vaccines to compare it to protections induced by other vaccine platforms.

Similarly to our results presented here, none of the previously tested vaccines based on NS proteins of ZIKV and YFV induced sterilizing protection against viral challenge in mouse models [40,43,44,46]. Although such protection was observed for vaccines based on surface proteins of ZIKV and YFV [3,4,42], this was most likely due to direct blocking of incoming virus particles by neutralizing antibodies binding to the surface proteins. Our group previously developed and evaluated a LV-based vaccine candidate encoding PrM and E proteins of ZIKV that induced sterilizing protection of *ifnar*−/− mice against lethal ZIKV challenge by inducing robust titre of neutralizing antibodies [49]. Conversely, vaccines targeting NS proteins mainly elicit T-cell responses, not directly neutralizing incoming virus particles but rather drastically limiting viral replication by killing virus-infected cells.

As wild type mice exhibit resistance to infections caused by many flaviviruses, the protective efficacy of vaccines is commonly evaluated in mice with permanent or temporarily induced immunodeficiencies. While such models are useful, immunodeficiencies could limit the magnitude of antiviral immune responses and underestimate the protective potential. Furthermore, since mouse and human T-cell responses do not target the same epitopes, the protection provided by the antigens designed to induce optimal T-cell response in humans can be underestimated in mouse models. Therefore, more relevant preclinical models

including non-human primates are needed to better evaluate the protective potential of such vaccines in humans.

### CRediT authorship contribution statement

Pierre Authié: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Philippe Souque: Validation, Methodology, Investigation, Formal analysis, Data curation. Fanny Moncoq: Validation, Methodology, Investigation, Data curation. Amandine Noirat: Methodology, Investigation, Formal analysis, Data curation. Catherine Blanc: Methodology, Investigation, Formal analysis, Data curation. Sylvain Ciret: Methodology, Investigation, Formal analysis, Data curation. Maryline Bourgine: Writing – review & editing, Validation, Resources, Data curation, Conceptualization. David Hardy: Visualization, Validation, Resources, Methodology, Formal analysis, Data curation. Françoise Guinet: Writing – original draft, Visualization, Validation, Supervision, Formal analysis, Data curation. Laleh Majlessi: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Data curation. Pierre Charneau: Writing – review & editing, Validation, Supervision, Resources, Methodology, Funding acquisition, Data curation, Conceptualization. Kirill Nemirov: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

### Funding

This work was funded by TheraVectys and Institut Pasteur.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Kirill Nemirov reports financial support, administrative support, article publishing charges, equipment, drugs, or supplies, and travel were provided by TheraVectys. Pierre Authie reports financial support, administrative support, article publishing charges, equipment, drugs, or supplies, and travel were provided by TheraVectys. Fanny Moncoq reports financial support, administrative support, article publishing charges, equipment, drugs, or supplies, and travel were provided by TheraVectys. Amandine Noirat reports financial support, administrative support, article publishing charges, equipment, drugs, or supplies, and travel were provided by TheraVectys. Laleh Majlessi reports financial support, administrative support, article publishing charges, equipment, drugs, or supplies, and travel were provided by TheraVectys. Pierre Charneau reports financial support, administrative support, article publishing charges, equipment, drugs, or supplies, and travel were provided by TheraVectys. Philippe Souque reports article publishing charges and equipment, drugs, or supplies were provided by TheraVectys. Catherine Blanc reports article publishing charges and equipment, drugs, or supplies were provided by TheraVectys. Sylvain Ciret reports article publishing charges and equipment, drugs, or supplies were provided by TheraVectys. Maryline Bourgine reports article publishing charges and equipment, drugs, or supplies were provided by TheraVectys. Kirill Nemirov reports a relationship with TheraVectys that includes: employment, funding grants, non-financial support, and travel reimbursement. Pierre Authie reports a relationship with TheraVectys that includes: employment, funding grants, non-financial support, and travel reimbursement. Fanny Moncoq reports a relationship with TheraVectys that includes: employment, funding grants, non-financial support, and travel reimbursement. Amandine Noirat reports a relationship with TheraVectys that includes: employment, funding grants, non-financial support, and travel reimbursement. Laleh Majlessi reports a relationship with TheraVectys that includes: consulting or advisory, funding grants, and non-financial support. Pierre Charneau
reports a relationship with TheraVects that includes: board member-
ship, consulting or advisory, equity or stocks, funding grants, and non-
financial support. Philippe Souque reports a relationship with Thera-
Vects that includes: funding grants. Catherine Blanc reports a rela-
tionship with TheraVects that includes: funding grants. Sylvain Ciret
reports a relationship with TheraVects that includes: funding grants.
Maryline Bourgine reports a relationship with TheraVects that in-
cludes: funding grants. Kirill Nemirov, Pierre Authie, Philippe Souque,
Amandine Noirat, Fanny Moncoq, Pierre Charneau has patent #WO
2024/084041 A2 pending to Assignee. PC is the founder and CSO of
TheraVects. PA, FM, AN, and KN are employed by TheraVects. LM has
a consultancy activity for TheraVects. PA, PS, FM, AN, PC, and KN are
inventors of a pending patent directed to the development of polyvalent
LV vaccine against DENV, Zika and yellow fever viruses. Other authors
declare no competing interests. If there are other authors, they declare
that they have no known competing financial interests or personal re-
lationships that could have appeared to influence the work reported in
this paper.

**Appendix A. Supplementary Data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.vaccine.2025.127563.

Data availability

Data will be made available on request.

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