key: cord-0291714-7bgx4ujb
authors: López, M. Verónica; Vinzón, Sabrina E.; Cafferata, Eduardo G. A.; Nuñez, Felipe J.; Soto, Ariadna; Sanchez-Lamas, Maximiliano; Afonso, Jimena; Aguilar-Cortes, Diana; Ríos, Gregorio D.; Maricato, Juliana T.; Torres-Braconi, Carla; Silveira, Vanessa Barbosa-da; Montes-de Andrade, Tatiane; Carvalho-de Souza Bonetti, Tatiana; Ramos Janini, Luiz M.; Castello Girão, Manoel J. B.; Llera, Andrea S.; Gomez, Karina; Ortega, Hugo H.; Berguer, Paula M.; Podhajcer, Osvaldo L.
title: A single shot of a hybrid hAdV5-based anti-COVID-19 vaccine induces a long-lasting immune response and broad coverage against VOC
date: 2021-08-11
journal: bioRxiv
DOI: 10.1101/2021.08.11.455942
sha: 6a0b3ad80c21e2d642236af231fe4f6de3e04c38
doc_id: 291714
cord_uid: 7bgx4ujb

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric hAdV5 vector. The vaccine (named CoroVaxG.3) is based on three pillars: i) high expression of Spike to enhance its immunodominance by using a potent promoter and a mRNA stabilizer; ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; iii) use of Spike stabilized in a prefusion conformation. Transduction with CoroVaxG.3 expressing Spike (D614G) dramatically enhanced Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3 vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha) and P.1 (gamma) Spikes, as well as an authentic WT and P.1 SARS-CoV-2 isolates. Neutralizing antibodies did not wane even after 5 months making this kind of vaccine a likely candidate to enter clinical trials

*Correspondence: opodhajcer@leloir.org.ar

The disease caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had an effect of enormous proportions, globally leading to more than 200 million confirmed cases and causing more than 4 million deaths (https://covid19.who.int/). During the last months different regulatory bodies issued emergency use authorization for different vaccines based mainly on mRNA and adenoviral-based platforms 1 . With the sole exception of the hAdV-26 based vaccine that was approved for one dose administration after showing an efficacy in clinical trials slightly over 65 % 2 , all the vaccines are given as a prime-boost approach to achieve maximal immune response and protection 3 . Companies are facing challenges in manufacturing vaccines and building the supply chains to meet the demand for COVID-19 vaccines. Different countries prioritized distribution of a first vaccine dose to as many people as possible 4 . Thus, the need for two doses and the fact that mRNA vaccines require logistically difficult cold-chains 5 make these type of vaccines more challenging to deploy in developing countries where ultra-low freezers may not be widely available. Therefore, getting a single dose vaccine with suitable stability and storage properties that can reach rapidly the local population is a major challenge for the scientific community especially in low and middle income countries.

Despite the unprecedented achievement of having approved vaccines in one year it is still early to establish the durability and extent of the protection, and recent data on the vaccines which first received approval, like BNT162b2 from Pfizer-BioNTech, point to a diminished efficacy already six months after vaccination 6, 7 . Most importantly, it is still unclear the way to optimize the existing vaccines to protect against the prevalent SARS-CoV-2 variants of concern (VOC) that are spreading globally 8 . These VOC have multiple mutations in Spike, mainly in the RBM region and N-terminal domain, that differ substantially from the Spike variants encoded by the already approved vaccines.

In fact, all the evidence indicates that serum samples obtained from convalescent people or from vaccines offer diminished protection against the β, γ and δ variants 9 .

Replication incompetent adenoviral-based vaccines can induce a long term adaptive neutralizing humoral and cellular immunity 10 . The prevalence of anti-hAdV5 immunity in the developing world in the past 11 led different academic groups and companies to identify less prevalent human adenovirus serotypes 2, 12 or non-human primates adenoviral vectors 13 for anti-COVID-19 vaccine development. However, previous studies that compared the immunogenicity induced by hAdV5-based vaccines with the less prevalent human adenovirus hAdV26 and the chimpanzee-derived adenovirus ChAdOx1 among others, demonstrated that hAdV5 induced the most potent immune responses 10, 14, 15 . Moreover, hAdV5 is no longer the most prevalent AdV responsible for pediatric and crowded community outbreaks and was replaced by other hAdVs 16 . In addition, there is preclinical evidence that the annual immunization with the same hAdV vector may be effective due to a significant decline in vector immunity 17 . Real world evidence also shows that hAdV-specific T cell response declines with age 18 .

Moreover, the clinical data obtained with a hAdV5-based COVID-19 vaccine showed that despite the pre-existence of hAdV5-nAbs, 85%-100% of volunteers administered with only one shot of a hAd5V-based vaccine showed seroconversion against SARS-CoV-2 19 . In a two dose regimen clinical trial with the ChAdOx1-based vaccine, anti-ChAdOx1 nAbs increased with the prime vaccination but not with the boost one; that was in contrast to anti-SARS-CoV-2 nAbs that continued to increase after the boost at 28 days 20 .

We designed a novel vaccine with the aim to enhance the immunodominance of the transgene. The vaccine, named CoroVaxG.3, is based on a replication incompetent hybrid hAdV5 where the expression of a prefusion stabilized full length Spike is transcriptionally regulated by a strong promoter and an mRNA stabilizer. We also engineered the hAdV5 vector-based vaccine to display the knob domain of hAdV3 in order to improve vector targeting to muscle and dendritic cells 21, 22 . Here, we describe the in vitro and in vivo data that makes CoroVaxG.3 a promising candidate to enter clinical trials.

In order to induce Spike immunodominance, the first step in our vaccine design was to select the most appropriate promoter to transcriptionally regulate Spike expression.

Other anti-COVID 19 vaccines based on replication-incompetent AdV incorporated different alternatives of the early intermediate CMV promoter to drive Spike transcription 23-26 . However, it was shown that the CMV promoter can be silenced due to methylation, not only when it is incorporated in viral vectors that integrate to the host genome 27 , but also after in vivo administration of a replication-deficient adenoviral vector in the rat muscle 28 . After an extensive search in the literature, we decided to synthetize different versions of a hybrid promoter that included a CMV enhancer, the chicken β-actin promoter and a chimeric intron that contains the 5' splice donor of the chicken β-actin 5'UTR and the 3' splice acceptor of the minute virus of mice (MVM). This hybrid promoter was shown to drive gene expression in the rat CNS following in vivo transduction with AAV vectors 29 . We designed different versions that differed mainly in the size of the CMV enhancer and the β-actin promoter. In addition, we incorporated stop codons in the three open reading frames after an ATG codon 3' splice acceptor of MVM that could interfere with Spike ATG. Two initial versions of the hybrid promoters named Pr1 and Pr2 were cloned into the pShuttle(PS)-IXP-Luc vector. Luciferase expression driven by Pr2-Luc was around 24fold (P < 0.0001) higher than the control SV40 promoter, compared to only 1.6-fold increase over the SV40 promoter induced by Pr1-Luc (Fig. 1a) . Based on these data, we decided to move on with Pr2 for the further constructs design.

Next, we aimed to establish the adenoviral vector capacity to transduce target cells following fiber knob exchange. We initially transduced human rhabdomyosarcoma cells Hs 729T (as an example of muscle cells), and human monocytes THP-1, with replication-deficient adenoviruses hAdV5-Luc and the hybrid hAdV5.3-Luc expressing the fiber knob domain of hAdV3. Using luciferase as a surrogate marker, we observed almost 40-fold induction and more than 100-fold induction in Hs 729T and THP-1 cells, respectively, with hAdV5.3-Luc compared to hAdV5-Luc ( Fig. 1b and c) . Fig. 2a and d) . Moreover, CoroVaxG.3 was able to induce at least 6 times higher Spike expression in THP-1 monocytes compared to CoroVaxG.5 ( Fig. 2b and e) .

Interestingly, CoroVaxG.3 induced a higher increase in Spike expression in THP-1 cells induced to differentiate to immature dendritic cells compared to undifferentiated THP-1 monocytes ( Fig. 2c and f) . Of note, we were unable to detect Spike expression in iDCs after cells transduction with CoroVaxG.5 even after membrane overexposure ( Fig. 2e and f).

Next, we aimed to establish whether fiber exchange might have affected the in vivo To reduce the theoretical risk of vaccine-associated enhanced respiratory disease, associated with a T H 2-skewed response 31 we further assessed the concentration of Sspecific IgG1 and IgG2a subclasses induced by CoroVaxG.3 as a readout of T H 1 and T H 2 responses. As a comparator we used sera from mice vaccinated either with CoroVaxG.5 or with the SARS-CoV2-S RBD domain in Freund's adjuvant (FA). RBD + FA vaccinated mice showed a low IgG2a/IgG1 ratio consistent with the production of high levels of IgG1 and a clear skew towards a T H 2 phenotype (Fig. 3c and d) . Interestingly, using sera samples obtained at day 28, we observed that CoroVaxG.3 and CoroVaxG.5 induced a high IgG2a/IgG1 ratio indicative of a T H 1-biased response, although the IgG2a/IgG1 ratio was slightly higher for CoroVaxG.5 than for CoroVaxG.3 ( Fig. 3d , P < 0.001). This difference was mainly due to a higher production of IgG1 by CoroVaxG.3, with similar levels of IgG2a induced by both vaccine candidates and doses (Fig. 3c) .

Similar data was obtained with sera collected 14 days after vaccination (not shown).

The generation and persistence of memory T cells provides life-long protection against pathogens and, in particular, the induction of virus-specific CD8+ T cell responses has the potential to improve the efficacy of vaccination strategies 32, 33 . In order to characterize the cellular immune response induced by a single immunization with CoroVaxG.3, we assessed IFN-γ production by isolated splenocytes after specific ex vivo re-stimulation with Spike peptide pools, using again CoroVaxG.5 as a comparator.

We observed an early strong primary immune response in vaccinated mice at 14 days post immunization, showing a similar range of IFN-γ secreting cells in both vaccinated groups (Fig. 4a) . Remarkably, IFN-γ secretion was induced up to 140 days following vaccination, with no significant differences observed between the two CoroVaxG vaccines (Fig. 4b) . Administration of Ad.C vector did not induce IFN-γ production at any of the assessed time points (Fig. 4a and b ).

The identification of distinct memory T cells is usually based on the differential cell surface expression levels of CD44 and CD62L. Effector-memory T cells (T EM ) located in secondary lymphoid organs can be identified as CD44 high CD62L low while spleen located central-memory T cells (T CM ) are identified as CD44 high CD62L high 34-37 . We observed a remarkable induction of CD44 high CD62L high CD8 + cells in splenocytes of vaccinated groups compared to control groups at day 140 after a single shot vaccination ( Fig. 4c and d). The proportion of CD44 high CD62L low CD8 + did not change in vaccinated mice at this time point (not shown). Ex vivo stimulated splenocytes in the Ad.C group showed levels of CD8 + cells expressing CD44 high CD62L high similar to those observed in naïve mice or unstimulated splenocytes. Collectively, the data show that mice vaccinated with both vaccines developed an effective long lasting memory T-cell response against SARS-CoV-2.

To evaluate the functional quality of vaccine-generated Spike-specific antibodies, we used a pseudovirion-based neutralization assay (PBNA) to test the ability of sera from immunized mice to neutralize the entry of pseudovirus bearing Spike on their surface. Sera collected from mice at days 14, 28 and 140 after vaccination, were tested for the presence of SARS-CoV-2-specific neutralizing antibodies (nAbs) (Fig. 5a ). NAbs against SARS-CoV-2 were detected in mice immunized by both CoroVaxG.3 and CoroVaxG.5.

The resulting SARS-CoV-2-neutralizing activity at all-time points assayed was statistically significant (P < 0.0001). compared to the undetectable nAbs in the Ad.C control group, with no significant differences between the vaccines. Remarkably, this neutralizing effect was observed even after 140 days of the single dose vaccination (Fig. 5b) . Interestingly, all sera tested neutralized the pseudovirus variants with IC 50 s of at least 150. Assessment of the same sera samples from day 28 used for the PBNA studies, confirmed that CoroVaxG.3 was able to elicit neutralizing antibodies against the authentic P.1/gamma VOC (Fig. 5c) .

In the present study we provide evidence on a novel, replication-deficient hAdV-based vaccine, aimed at achieving Spike immunodominance to skew the immune response towards the transgene, and reducing the vector-targeted immune response that will occur despite the vector used. Immunodominance of Spike was tackled from different angles: (i) optimizing Spike expression using a potent promoter and an mRNA stabilizer; (ii) stabilizing Spike in a prefusion conformation to enhance Spike immunogenicity 38, 39 ; (iii) engineering the hAdV5 fiber to express the fiber knob domain of hAdV3 to enhance transduction of muscle and dendritic cells.

We observed a dramatic enhancement of Spike expression in human muscle cells as well as in human monocytes and dendritic cells with CoroVaxG.3 compared to CoroVaxG.5. Since in both vaccine candidates Spike expression was under the regulation of Pr2 and the WPRE mRNA stabilizer, we concluded that the enhanced Spike expression in human muscle derived cells, monocytes and dendritic cells was due to the fiber knob exchange as demonstrated also through the studies using replicationdeficient vectors expressing luciferase. One of the main initial concerns regarding vaccine development was the possibility of vaccine-related diseases, either induced by antibodies (antibody-dependent enhanced disease, ADE), which is associated with the presence of non-neutralizing antibodies, or vaccine-associated enhanced respiratory disease (VAERD), linked to inflammation induced by Th2-skewed immune responses 44, 45 . On the other hand, the induction of an unbalanced inflammatory Th1-biased response by some Ad vectors, due to an exacerbated production of type I interferons, has been suggested to impair transgene expression levels, dampening subsequent humoral immune responses 46 . Therefore, the induction of a balanced immune response is desirable. Interestingly and despite the fact that both vaccines candidates induced a robust and long lasting humoral and cellular immune response, CoroVaxG.3 elicited higher levels of IgG1 and hence a more balanced Th1/Th2 ratio, evidencing an optimal immune profile in mice. The difference in the subclass profile between the two vaccines might be related to the differential tropism observed in the in vitro studies. It was already shown that mice injected with DCs transduced ex vivo with hAdV5 expressing βgal elicited mainly IgG2a antibodies, while vaccination with hAdV5-βgal transduced myoblasts elicited a more balanced Ab response with an IgG1/IgG2a ratio similar to that induced by direct vaccination with hAdV5-βgal 47 ; thus, it is likely that CoroVaxG.3 is inducing a more balanced immune response due to its enhanced tropism for muscle cells.

In acute and convalescent COVID-19 patients it has been observed that the presence of T cell responses is associated with reduced disease [48] [49] [50] , suggesting that SARS-CoV-2specific T cell responses may be important for control and resolution of primary SARS-CoV-2 infection 51 . Preclinical studies in macaques have shown that both neutralizing antibody titers and Fc functional antibody responses correlated with protection 52, 53 and that purified IgG from convalescent animals, in the absence of cellular and innate immunity, effectively protected naïve recipients against a challenge with SARS-CoV-2 54 .

However, in the setting of waning and subprotective antibody titers, cellular immune responses were critical for rapid virological control. Through CD8 depletion studies, it was shown that cellular immunity, especially CD8+ T cells, contributed to protection against rechallenge with SARS-CoV-2 51 A correlate of protection that allows evaluation of vaccine efficacy based on immune readouts has been sought since the beginning of COVID-19 vaccine development.

Recent studies suggest that neutralizing antibodies could serve this purpose and are, therefore, the main parameter that might help to predict the success of a vaccine candidate 58 . CoroVaxG.3 was able to elicit neutralizing antibodies to a similar extent to the levels previously reported in preclinical animal models, and those nAbs were stable in the long term. Moreover, studies with pseudoviruses and authentic SARS-CoV-2 containing variant substitutions suggested that neutralizing ability of the antibodies in sera raised after CoroVaxG.3 vaccination is only slightly reduced but overall largely preserved against the B.1.1.7 and P.1 lineages that are VOC prevalent in Latin America 59 . This mild reduction in neutralization by vaccine-raised sera against these VOC is in the same range than that observed for sera of patients who received two doses of either the BNT162b2 Pfizer-BioNTech or ChAdOx1 nCoV-19 Oxford-AstraZeneca vaccine 60 .

Each one of the AdV-based vaccines already approved by different regulatory bodies and the CoroVaxG.3 platform described in this study are based on different AdV species or human serotypes, are designed in a different way and most importantly, bind to different cell surface receptors 10 . Although some adenoviral vectors from human and non-human primate origin display robust immunogenicity in vivo comparable to that of AdV5, they are less immunogenic 61, 62 , and there are considerable differences in the phenotype and functionality of the immune response they can elicit 15, 43, 61 . These differences could clearly impact on the durability, potency and quality of the immune response induced by the different adenoviral-based vaccines. Thus, it is likely to assume that they might trigger a differential immune response that will not necessarily be visible during the short time that evolved since immunization with the different vaccines started, and might require an extended follow-up period to emerge. How the different immune profiles will influence vaccine efficacy in patients remains to be seen. We developed a novel anti-COVID-19 vaccine based on a hybrid hAdV5 vector that expresses a chimeric fiber and where the high expression of Spike stabilized in its prefusion state is supportive to achieve its in vivo immunodominance. CoroVaxG.3 showed differential characteristics compared to CoroVaxG.5, a vaccine comparator with features similar to many vaccines that gained regulatory approval. Our study demonstrated that the high levels of neutralizing antibodies elicited by CoroVaxG.3 were maintained for at least 5 months, and probably much longer, since they were stable over the analyzed period. Moreover, sera obtained from CoroVaxG.3 vaccinated mice, were able to neutralize VOC of regional

importance. An anti-COVID 19 vaccine based on the molecular design of CoroVaxG.3 is ready to enter clinical trials in the next coming months based on a one shot administration. HEK293T-hACE2 cells were already described 64 . All the cell lines were grown in the recommended medium supplemented with 15% of fetal bovine serum (Natocor, Cordoba, Argentina), 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin and maintained in a 37 °C atmosphere containing 5% CO 2 . For HEK293T and HEK293T-hACE2 cell cultures non-essential amino acids (1X final concentration) were added. Immature dendritic cells (iDC) were generated from THP-1 monocytes as previously described 65 hAdV5/3-Luc and hAdV5-Luc replication-deficient adenoviral vectors were already described 63 . Hs 729T and THP-1 cells were transduced with hAdV5-Luc and hAdV5/3-Luc viruses at MOI 500. Forty-eight hours later, the cells were collected and assayed for Renilla Luciferase activity as described.

The sequence of the Spike protein gene was extracted from the official GISAID reference sequence WIV04 (https://www.gisaid.org/) and modified to obtain the 

For IgG1 and IgG2a ELISAs, plates were coated with SARS-CoV-2 Spike protein as described in the previous section. The S-specific IgG1e3 and IgG2a mAbs (Invivogen) were serially diluted from 200 ng/mL to 3.125 ng/mL in blocking buffer and incubated 1 h at room temperature. Mouse sera were diluted 1:150 or 1:1500 in blocking buffer in order to fit the linear range of the standard curve. After the plates were washed, HRP-conjugated goat anti-mouse IgG1 and IgG2a (1:20000, ab97240 and ab97245, Abcam) were added to each well and the ELISA was performed as described before.

For each IgG subclass reference, a standard curve was plotted using GraphPad Prism 8.0 generating a four-parameter logistical (4PL) fit of the OD 450 nm at each serial antibody dilution. In this way, the relative levels were comparable between IgG subclasses measured on the same antigen. As a comparator, the sera from 3 mice inoculated with RBD + Freund's adjuvant was included.

The pseudoviral particles (PVs) containing SARS-CoV2 Spike-D614G protein were generated according to the methodology described by Nie et al 66 After 48 hours the supernatant containing the PVs was collected, filtered (0.45-μm pore size, Millipore) and stored in single-use aliquots at -80 °C. The 50% tissue culture infectious dose (TCID 50 ) of SARS-CoV-2 PV was determined in sextuplicates and calculated using the Reed-Muench method as previously described 66 .

The neutralization assays were performed as previously described 66 

Neutralizing antibody (nAb) titers against SARS-CoV2 were defined according to the 

All quantitative data are presented as the means ± SEM. ANOVA and two-sample t tests were used to compare continuous outcomes between groups. Differences were considered significant if P < 0.05. The statistical tests used are indicated in each figure legend. For S-specific binding antibodies as measured by ELISA (Fig. 3b) and PnAb titers as measured by PBNA (Fig. 5a) , data were log-10 transformed prior to statistical analysis. Statistical differences between immunization regimens and time points after immunization were evaluated two-sided using a two-way ANOVA and a multiple comparisons Bonferroni correction was performed a posteriori. To compare isotype ratios between treatments data were log-10 transformed and a one-way ANOVA Brown-Forsythe test was applied. The change in neutralization for the different VOC was assessed by a Wilcoxon matched-pairs signed rank test. Comparison between the vaccinated and naïve sera in the VNT was done applying a two-tailed Mann-Whitney U test. All analyses were conducted using GraphPad Prism software (version 8.2). All statistical tests were conducted two-sided at an overall significance level of α = 0.05. Changes in reciprocal serum neutralization IC 50 values of CoroVaxG.3 vaccinated mouse sera (28 days post vaccination) against SARS-CoV-2 variants of concern. Fold change in Geometric Mean Titers relative to the WT is written above the p values.

Statistical analysis was performed using a Wilcoxon matched-pairs signed rank test. 

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Sponsored Research Agreement of Fundacion Instituto Leloir-CONICET(IIBBA) with Vaxinz AUTHOR CONTRIBUTIONS Design of vaccines

Contributed resources

The authors declare no competing financial interests. O.L.P., M.V.L., S.E.V., E.G.C. and F.J.N. are co-inventors on a related vaccine patent.