Self-Amplifying RNAs Continue to Change the Game in Vaccine Development

Introduction??????

Self-amplifying RNA (saRNA) is of interest for lower dosing and more durable biological effects compared to conventional mRNA. As discussed in an earlier review -, these attractive features of saRNA are due to its ability to generate multiple copies of RNA-encoded protein antigens or therapeutic agents. An overview of saRNA for vaccines and cancer therapies was provided in a follow-up blog at the beginning of 2023. Since then, there have been more than 35 publications on saRNA added to the NIH PubMed database, all of which can be accessed at this link. ?

Among these saRNA publications in 2023, the following four vaccine-related studies are featured in the present blog, and include the advantages of incorporating modified nucleosides in saRNA:?

1. World’s first saRNA vaccine approved in Japan.?

2. Clinical comparison of a nucleoside-unmodified saRNA to the nucleoside-modified (N1-methylpseudouridine, m1ψ) Pfizer/BioNTech mRNA COVID-19 vaccine.?

3.Clinical comparison of a nucleoside-modified (5-methylcytosine, m5C) saRNA with its nucleoside-unmodified version as COVID-19 vaccines.?

4. Complete substitution of saRNA with certain modified nucleosides suppresses early interferon response and increases potency in vivo.??

1. World’s first saRNA vaccine approved in Japan

On November 27, 2023, Japanese regulators?authorized?ARCT-154, a new COVID-19 vaccine created by Arcturus Therapeutics (a California-based vaccine developer) and CSL (an Australian biotech firm), making it the first fully approved?COVID-19 vaccine. This liposome nanoparticle (LNP)-formulated saRNA -was approved as both an initial COVID-19 vaccine and a booster for people over the age of 18. ?

Japan based its approval on data from several trials, including one in which more than 800 people received either a 5-μg dose of the saRNA shot or a 30-μg dose of Pfizer/BioNTech’s COVID-19 mRNA vaccine as a booster. According to a?preprint, those who received the ARCT-154 booster had higher levels of COVID-neutralizing antibodies, while side effects were about equal between the two groups.?

In another preprint of an ongoing study involving 16,000 people in Vietnam, ARCT-154 saRNA is reported to be based upon the Venezuelan Equine Encephalitis Virus (VEEV) replicon expression vector in which RNA coding for the virus structural proteins has been replaced with RNA coding for the full-length Spike glycoprotein of the SARS-CoV-2 D614G virus, an early variant of the ancestral strain containing a single point mutation.??

It found that two 5-μg doses, delivered 28 days apart, are 56% effective at preventing COVID-19 and 95% effective against?COVID-19. Most cases at the time of this study were due to the Delta variant, and side effects were mild to moderate.?

Arcturus and CSL will now focus on getting the shot approved by European authorities — that decision is?expected in 2024.?

2. Nucleoside-unmodified saRNA m1ψ-modified mRNA as COVID-19 vaccines

Another nucleoside-unmodified VEEV-based saRNA COVID-19 vaccine also encoding a SARS-CoV-2 Spike protein was reported by Komori et al. (2023).This preclinical study of non-human primates demonstrated the formation of high levels of neutralizing antibodies for at least 12 months.??

A follow-up Phase 1 clinical study compared this saRNA, termed VLPCOV-01, with the m1ψ-modified Pfizer/BioNTech BNT162b2 mRNA vaccine. Ninety-six participants, who had previously received two doses of the BNT162b2 mRNA vaccine, were evenly divided into two age groups, 18-55 years and ≥65 years, each randomized to receive one injection of 0.3, 1.0, or 3.0 μg of VLPCOV-01, or 30 μg of BNT162b2, or placebo.??

VLPCOV-01 induced robust virus-specific immunoglobulin G (IgG) titers that were maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5,000 at 0.3 μg to 13,000 at 3 μg, compared to only 3,000 for 30 μg of BNT162b2. Notably, these data indicate ?10-fold higher potency of VLPCOV-01 vs. BNT162b2, a finding also reflected in the elderly cohort IgG titers at 26 weeks post-vaccination.??

On the other hand, the undesired reactogenicity of VLPCOV-01 was comparable to that of BNT162b2, notwithstanding its 10-fold lower dose. Nucleoside-unmodified saRNA VLPCOV-01 was said to be more prone to degradation compared to m1ψ-modified BNT162b2 mRNA, which may have contributed to the level of reactogenicity of VLPCOV-01. To investigate this important possibility, the following study was carried out.??

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3. Nucleoside-modified m5C saRNA nucleoside-unmodified saRNA vaccines

According to Aboshi et al. (2023), reactogenicity is reduced by incorporating m1ψ in Pfizer/BioNTech BNT162b2 mRNA; however, m1ψ in saRNA inhibits VEEV RNA-dependent RNA polymerase or transcriptase function. Therefore, the possibility of suppressing saRNA VLPCOV-01 reactogenicity was explored by incorporation of m5C, by analogy to what is known for the effect of m5C on mRNA (Karikó et al. 2005). This m5C-modified variant of VLPCOV-01 was termed VLPCOV-02.?

Like the Phase 1 study described above for VLPCOV-01, 96 previously vaccinated participants were equally divided into non-elderly and elderly cohorts and were randomized to receive a single booster vaccination with VLPCOV-02 at one of four doses, 1.0, 3.0, 7.5, or 15 μg. Geometric mean IgG titers increased between day 0 and day 28 at each dose level:

2 to 5-fold in the non-elderly cohort and 2 to 3-fold in the elderly cohort, indicating that m5C-modified VLPCOV-2 is immunogenic.?

Body temperature was used as a measure of reactogenicity. The body temperature of all participants who received VLPCOV-02 peaked within 24 hours post-immunization, regardless of age cohort or dose of vaccine, and returned to normal within 1 day in all but one participant, in whom it lasted 2 days. The frequency and magnitude of rise in body temperature at 24 hours increased in a dose-dependent manner and were particularly low in the groups who received lower vaccine doses, 1.0 and 3.0 μg. ?

Importantly, VLPCOV-02 was less reactogenic than VLPCOV-01, based on rates of fever in participants receiving the same dose of vaccine. Among the 20 participants who received 3.0 μg of VLPCOV-01, 30% experienced fever while, in the present study, among the 24 participants who received 3.0 μg of VLPCOV-02, only 4% experienced fever.??

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4. Complete substitution of saRNA with certain modified nucleosides suppresses early interferon response and increases potency in vivo.

In vitro co-transcription with CleanCap? AU analog and nucleoside-modified NTPs from TriLink was used by McGee et al. (2023) to synthesize 26 different mCherry reporter-saRNAs wherein a modified nucleoside completely replaced its unmodified counterpart. These 26 modified nucleosides were comprised of 6 A, 6 C, 4 G, 5 U and 5 ψ analogs.?

LNP-mediated transfection these 26 mCherry saRNAs into a T-cell line led to identification of 3 modifications that afforded significantly elevated levels of mCherry-positive cells by FACS, compared to the unmodified control, namely, 5-hydroxymethylcytidine (5OHmC), 14-fold; m5C, 10-fold; and 5-methyluridine (m5U), 8-fold.??

This better performance with m5C was also found in vivo. An injection of mice with 2.5 μg of LNP-formulated m5C-saRNA encoding firefly luciferase showed? 4-fold higher bioluminescence compared to m1ψ-modified mRNA after 7 days, and persisted at this increased level up to 28 days.?

It is known that, without modified nucleosides, exogenous RNA activates toll-like receptors and triggers the production of type I interferons (IFNs), resulting in translational shutoff and systemic inflammation. To compare the levels of IFNs induced by nucleoside-modified mCherry saRNAs, cultured human peripheral blood mononuclear cells from three donors were transfected. Gene expression analysis revealed that unmodified saRNA treatment induces a significant increase in the expression of early IFN-related genes after 6 hours. However, incorporation of 5OHmC or m5C led to a large reduction in the expression of these early IFN genes.?

Encouraged by these findings, m5C-modified and unmodified saRNAs encoding SARS-CoV-2 Spike protein were prepared. In a SARS-CoV-2 challenge study, a group of mice receiving a 10-ng dose of the unmodified saRNA showed severe weight loss and 50% lethality at 12 days post-infection. In contrast, a group of mice vaccinated with a 10-ng dose of the m5C-modified saRNA demonstrated weight-gain and only 10% lethality, which was a statistically significant (p < 0.05) improvement in survival.??

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Concluding Comments?

The approval of ARCT-154 saRNA as a vaccine for COVID-19 represents a significant milestone for saRNA-based vaccinology inasmuch as it the first instance of successful development of any saRNA from “design-to-drug.” In addition, the 6-fold lower efficacious dose for ARCT-154 compared to Pfizer/BioNTech’s COVID-19 mRNA vaccine as a booster with comparable safety provides support for the anticipated potency advantage of saRNA vs. conventional mRNA.?

Finding that incorporation of 5OHmC, m5C, or m5U in saRNA can suppress undesired reactogenicity/early IFN responses and afford increased yields of encoded protein/potency is also particularly noteworthy. These and other types of “masked” yet potent nucleoside-modified saRNAs could be advantageously applied to other vaccines and therapeutics, thus making modified saRNA an attractive alternative to conventional modified mRNA.?

These newly found advantages of incorporating modified nucleosides into saRNA underscore the significance of the 2023 Nobel Prize awarded to Katalin Karikó and Drew Weissman for their discoveries concerning nucleoside base modifications for enabling the development of effective mRNA vaccines against COVID-19.?