key: cord-0312819-5tv2piyq
authors: Flynn, Olivia; Dillane, Kate; Lanza, Juliane Sousa; Marshall, Jennifer M.; Jin, Jing; Silk, Sarah E.; Draper, Simon J.; Moore, Anne C.
title: Low adenovirus vaccine doses administered to skin using microneedle patches induce better functional antibody immunogenicity as compared to systemic injection
date: 2021-03-16
journal: bioRxiv
DOI: 10.1101/2021.01.21.427553
sha: b86e65d866cd3110078020ce47512fbd1628b1c7
doc_id: 312819
cord_uid: 5tv2piyq

Adenovirus-based vaccines are demonstrating promising clinical potential for multiple infectious diseases including COVID-19. However the immunogenicity of the vector itself decreases its effectiveness as a boosting vaccine due to the induction of strong anti-vector neutralising immunity. Here we determined how dissolvable microneedle patches (DMN) for skin immunization can overcome this issue, using a clinically-relevant adenovirus-based Plasmodium falciparum malaria vaccine, AdHu5-PfRH5, in mice. Incorporation of vaccine into patches significantly enhanced its thermostability compared to the liquid form. Conventional high dose repeated immunization by the intramuscular (IM) route induced low antigen-specific IgG titres and high anti-vector immunity. A low priming dose of vaccine, by the IM route but more so using DMN patches, induced the most efficacious immune responses, assessed by parasite growth inhibitory activity (GIA) assays. Administration of low dose AdHu5-PfRH5 using patches to the skin, boosted by high dose IM, induced the highest antigen-specific serum IgG response after boosting, the greatest skewing of the antibody response towards the antigen and away from the vector and the highest efficacy. This study therefore demonstrates that repeated use of the same adenovirus vaccine can be highly immunogenic towards the transgene if a low dose is used to prime the response. It also provides a method of stabilising adenovirus vaccine, in easy-to-administer dissolvable microneedle patches, permitting storage and distribution out of cold chain.

applicable simian adenoviruses, as previously demonstrated 22, 23 .

To date, administration of adenovirus vaccines by microneedle patches in mice has 49 focussed on the induction of T cell responses to HIV antigens 24, 25 and there is no 50 understanding of the effect of anti-vector immunity or the induction of functional/protective 51 humoral immunity using DMN patches in any model. In this study, we address this problem 52 and examine the induction of vector-specific and functional antigen-specific antibody 53 responses using DMN patches fabricated using our simple process.

The development of highly effective and durable vaccines against the human malaria 55 parasites Plasmodium falciparum and P. vivax remains challenging 5, 26 . One vaccine strategy 56 is targeted at the blood-stage of the disease by blocking merozoite invasion of red blood 57 cells. This approach will likely require the maintenance of high titres of neutralizing 58 antibodies to merozoite proteins. Plasmodium falciparum reticulocyte-binding protein 59 homolog 5 (RH5) has emerged as a strong blood-stage vaccine candidate that is being 60 clinically tested. In parallel with this development, non-human primate studies have 61 demonstrated that the in vitro assay of antibody-mediated growth inhibition activity (GIA) 62 against P. falciparum serves as an in vitro correlate of vaccine-induced in vivo protection 63 when using vaccines targeting PfRH5 and other blood-stage antigens [27] [28] [29] .

The objective of this study was therefore to determine how the dose and method of delivery, either IM injection or skin administration using a DMN patch, of an adenovirus vectored 66 blood-stage malaria vaccine impacts on the induction of neutralising anti-parasitic antibodies 67 and anti-vector antibodies in mice.

We previously demonstrated short-term stability of an AdHu5 vector embedded in our DMN 71 patches and kept at ambient temperature over 14 days 15 . Here, we stressed the vaccine at 72 ambient and high temperature conditions for 6 months to better determine the vaccine's were stored at 25C and 60% relative humidity (RH) or 40C and 75% RH for up to 6 75 months, according to ICH Q1A guidelines 30 . Adenovirus infectivity was assessed in patches 76 or in liquid at 1 week or 1, 2, 3 and 6 months. Adenovirus stored in the liquid form at the 77 higher temperature, quickly degraded within a week and viable virus was undetectable at 78 one month (Fig 1A) . In contrast, no degradation of the vaccine in patches was observed at 79 40C for up to 2 months, however vaccine degradation was observed when patches were 80 stored at 40C for 3 months. No visible changes to the microneedles were observed in these 81 patches stored at 40C up to 6 months storage (Fig 1C) . At the accelerated temperature of 

We previously demonstrated influenza vaccine dose sparing using dissolvable microneedles 90 in mice 15 either IM ( 1 x 10 5 ifu) or skin ( 5 x 10 5 ifu) route, also induced significantly lower antiadenovirus IgG responses after the first immunization, compared to the standard 1 x 10 8 ifu All groups were boosted by the same IM route and dose, so that post-boost immunity only reflected differences induced in the primary immunization. All groups had a significantly higher IgG to PfRH5 post-boost, compared to the primary immunization (p < 0.05, paired tantigen-specific IgG responses as compared to the conventional IM immunization (Fig 2A) . In contrast, a high level of variability was observed in animals primed with low doses of 110 vaccine by the IM route. The IM high dose boost caused a significant increase in anti-vector 111 IgG in all groups as compared to the post-prime anti-vector response (Fig 2B) . A dose 112 response effect was observed with respect to anti-vector responses post-boost ( Fig 2B) .

anti-vector responses compared to the 1 x 10 8 ifu IM group.

To determine how anti-AdHu5 IgG antibodies reflected functional, adenovirus neutralising 116 capacity, titres of neutralising antibodies (NAb) were determined by hexon infectivity assay 117 for high and low vaccine doses by the IM and skin routes after boosting ( Fig 2D) . A dose 118 dependence was observed, irrespective of route, whereby low vaccine doses (1-5 x 10 4 ifu) 119 induced significantly weaker anti-adenovirus NAb titres as compared to priming with 1-5 x 120 10 7 ifu or with 1 x 10 8 ifu IM.

Using the antibody titres to the antigen (Fig 2A) and to the vector (Fig 2B) , we analysed the 122 skewing of the antibody response to the encoded antigen and away from the virus vector.

Conventional immunization, using 1 x 10 8 ifu AdHu5-PfRH5 by the IM route primed a 124 humoral response whereby the anti-transgene response was 3.5 times higher than the anti-125 vector antibody response, as evidenced by an equivalent or higher relative level of vector-126 specific antibodies compared to antigen-specific antibodies (geometric mean; 3.5, 95% CI; antigen-to vector-specific antibodies from prime to boost in the lower doses (1 x 10 5 or 1 x antibodies could not be modulated by the boost. In contrast, at the lowest vaccine dose 138 administered by DMN patches, the ratio of antigen to vector specific responses was significantly altered, and increased, from prime to boost; the geometric mean ratio postprime was 198 (95% CI: 131 to 299) and post-boost this significantly increased to a the IM route induced the highest ratio of antigen-specific to vector-specific antibodies; these 144 were significantly higher than the IM 1 x 10 8 ifu approach and this was the only prime-boost 145 approach that led to a significant increase in this ratio post-boost. Overall, these results 

These results demonstrate that priming and boosting with a low dose (5 x 10 4 ifu) of vaccine, using patches or by the IM route, significantly skews the antibody response towards the antigen and away from the vector, as compared to high dose administration.

immunization induced an antibody response that contained low levels of growth inhibitory antibodies (median 45%, 95% CI; 37% to 56%). A high dose of vaccine administered by 

Therefore, there is a balance between inducing the highest antigen-specific immunity (and 199 functional antibodies) and using the simplest, homologous immunization regime. 

We examined the antigen-specific IgG1 and IgG2a titres to determine if there were 203 significant differences in the ratio of these subtypes due to the vaccine dose and route of 204 immunization. Only groups that had high levels of GIA were examined and compared to the 205 conventional, high dose repeated IM immunization regimen. It was observed that IMlo/IM 206 10^8 and DMNlo/IM 10^8 significantly increased IgG1 titres compared to repeated high dose 207 IM (Fig 5A) . A repeated low dose of vaccine administered with patches (DMNlo/DMNlo) 208 induced significantly reduced antigen-specific IgG2a compared to repeated high dose vaccine by the IM route. Using a patch in the prime skewed the response away from IgG2a; 210 the ratio of IgG1:IgG2a was approximately 100-fold higher when vaccine was administered 211 using a patch in the prime and boost (Fig 5B) . 

Finally, we assessed the kinetics of the IgG response over time after a single immunization the DMN patch (Fig 6) . The antibody responses to PfRH5 remained relatively stable in most 

We also demonstrated short-term stability of an adenovirus virus vector in DMN patches 15 .

Here, we extended this finding to demonstrate the significant stability advantages of immunization, however it is unclear how strong this boosting effect was in comparison to IM 276 delivery. Therefore it is possible to boost weak antigen-specific serum antibody titres in 277 macaques by repeated Ad-DMN immunization, however the comparative magnitude of this effect to injected vaccine is unknown 25 . Our present study suggests that repeated low doses 279 or a low/high prime/boost, repeated adenovirus regime, could be a more suitable regimen to further increase the magnitude of these responses. We previously demonstrated that these 281 sugar-based microneedles fully dissolve in <10 minutes when administered to porcine skin 15 282 and therefore deliver vaccine to the body in a short timeframe, similar to the time period for 283 IM delivery. We observed a similar kinetic profile of the antibody response after a single 284 immunisation using an IM injection or a DMN patch (Fig. 6) . Therefore, although the 285 magnitude of the antibody responses were dependent on the dose and delivery system, the Female Balb/c mice were immunized on day 0 with AdHu5-PfRH5 by the IM route or using DMN patches. Mice were administered the standard 1 x 10 8 ifu by IM injection or with a DMN patch ("IM hi" or "DMN hi"). Alternatively, mice were administered a low dose of 5 x 10 4 ifu by IM injection or DMN patch ("IM lo" or "DMN lo").

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Symbols represent median, bars represent interquartile range. For IgG responses to PfRH5, no significant differences in titres were observed over time for IM-lo or DMN-hi

05 for day 56 compared to day 119

01; day 17 compared to day 119, by one-way ANOVA, Kruskal-Wallis test with Dunn's multiple comparison. For IgG responses to AdHu5

05 for day 17 compared to day 56 and day 119; in DMN-hi **,p < 0.01 for day 17 compared to day 56 and d119

001 compared to all other timepoints; no significant differences in titre were observed over time for DMN-lo, by one-way ANOVA, Kruskal-Wallis test with Dunn's multiple comparison

The authors would like to thank Dr. Conor O'Mahony, Tyndall National Institute, UCC for $$ $$