Oligonucleotide therapeutics breaking new ground – modified mRNA vaccines for ZIKA virus demonstrate rapid vaccine development for emerging diseases
Messenger RNA was first used as a vector to express proteins in mice in 1990 (1), but has not gained nearly the same attention as DNA vectors, largely because most researchers consider mRNA expensive to produce as well as too fragile and difficult to work with. However, growing experience over the last 10 years has demonstrated that these challenges can be addressed and mRNA therapeutics are starting to emerge as an exciting new class of oligonucleotide therapeutics with CureVac, BioNTech and more recently Moderna Therapeutics leading the field. (2) CureVac and BioNTech initially focused their mRNA platform on cancer immunization. However, clinical trials using mRNA vaccines against prostate cancer (NCT00831467), non-small-cell lung cancer (NCT01817738) and renal cell carcinoma (NCT01582672) have shown that although immune responses against cancer neo-antigens can be achieved, this does not necessarily translate into significant therapeutic benefit. It seems that these companies have recognised that this is not necessarily a failing of the mRNA technology itself, but the result of the complicated interplay between tumours and the immune system that makes it challenging to eradicate the disease. These companies seem to have realised that the “low-hanging fruit” of mRNA therapeutics is its use as vaccination technology against emerging and re-emerging infectious diseases.
In a world of highly efficient international transport and increased urban density particularly in countries with poor health care, it becomes increasingly difficult to contain emerging and re-emerging diseases at the point of origin. This has been abundantly demonstrated by a number of disease outbreaks such as HIV, SARS-coronavirus in 2002, the H1N1/09 influenza strain commonly known as swine flu in 2009, Ebola virus in 2013-2016 and most recently, ZIKA virus. Public health measures have had varying degrees of success in limiting the global impact and spread of these infectious diseases. Efforts to contain outbreaks are hampered by the fact that development and production of new vaccines for emerging pathogens takes years for traditional live attenuated or inactivated vaccines even under emergency response conditions. Simply adapting the well-established production process for the yearly influenza vaccine (an inactivated vaccine) to switch to a new strain takes a minimum of 20 weeks due to the required upscaling from a seed culture to full production capacity.
Such delays could make the difference between a fairly contained outbreak and a full-blown pandemic. With the number and diversity of disease outbreaks on the rise (3, 4), ways to accelerate vaccine development and manufacture are clearly needed.
One way to speed up these processes is the use of mRNA, DNA or recombinant viral vectors to express a particularly immunogenic antigen directly within cells. This should closely mimic an actual pathogenic infection and thus have the additional advantage of eliciting both strong B and T cell immune responses. Initially, DNA vaccines seemed to be the most promising avenue as production would be significantly simplified, easily scalable and could be switched over to a different DNA vaccine in a matter of days. Unfortunately, first-generation DNA vaccines were only poorly immunogenic due delivery issues, specifically the need for the DNA to enter the cell nucleus. (5) Consequently, attention has mostly focused on recombinant vector vaccines, with several candidates currently in clinical trials. However, the potential for insertional mutagenesis with both DNA and viral vector vaccines as well as pre-existing immunity to the vectors used in the latter are ongoing concerns.
Until recently, mRNA was thought to be the least appealing of these three options. Yet, mRNA vaccines would have significant advantages even over DNA vaccines: genomic integration is not possible, delivery into the cytoplasm is sufficient, the transient nature of antigen expression is unlikely to induce immune tolerance and mRNA vaccines work in non-dividing cells. Progress has also been made in optimizing delivery, mRNA stability and translation efficiency. For example, current modified mRNA vaccines are effectively delivered using lipid nanoparticles and contain nucleoside modifications such as 5-methylcytidine, pseudouridine or N(1)-methyl-pseudouridine. (6) Changing the mRNA cap 0 structure (m7GpppG) to a cap 1 (m7GpppNm) avoids activation of the innate immune response against foreign RNA via the receptor Retinoic Acid Inducible Gene-I (RIG-I) while adding 5′ and 3′ untranslated regions derived from known stable mRNAs increases stability. Optimizing the GC content and thus mRNA secondary structure by careful sequence engineering as well as adjusting the length of the poly (A) tail can also influence stability and translation efficiency (7). Addition of signal peptides to the vaccination antigen can enhance presentation and secretion of the antigen and thus increase immunogenicity.
These improvements combined with the undeniable advantages of mRNA vaccines have led to a resurgence of interest in this technology, particularly for developing a vaccine against ZIKA virus, the causative agent of the latest major outbreak. In the last three months, two papers showing preclinical data from mRNA vaccination against ZIKA virus in mice and non-human primates have been published. (8, 9)
First identified in humans in 1954 in Nigeria and 1966 in Malaysia, ZIKA virus is a mosquito-borne flavivirus that was thought to cause only mild disease with fever, malaise, muscle and joint pains, conjunctivitis, rash, anorexia and dizziness. (10) Evidence suggests that ZIKA is widespread in Afrika and Asia, but in 2007 it spread from Asia to Micronesia, then to French Polynesia in 2013 and to northern Brazil in late 2014. By 2016 the virus had spread to most of South and Central America. Worryingly, a 20-fold increase in Guillain-Barre syndrome (GBS), an autoimmune response that can temporarily or permanently damage the peripheral nervous system, had been noted during the French Polynesia outbreak. In late 2015, huge increases in newborns with microcephaly were associated with the ZIKA outbreak in northern Brazil and surrounding countries. It is as yet unclear if the virus underwent some form of genetic adaptation that lead to more efficient transmission or higher viremia levels and thus causes more severe disease or if this is simply due to the exposure of a naïve population without pre-existing herd immunity. (11) Another possible explanation is that pre-existing antibody responses to the closely related Dengue virus could enhance the severity of ZIKA virus infection. At the moment, Dengue is extremely prevalent in the northern parts of South America and in Central America and antibody-dependent enhancement (ADE) is well-established as the cause of severe Dengue disease (dengue haemorrhagic fever or dengue shock syndrome) after secondary infection with a heterotypic Dengue virus serotype. Mosquitos are able to transmit Zika and Dengue with the same bite (12) and, at least in mice, Dengue-mediated ADE has been shown to increase viremia and enhance mortality of ZIKA virus infection (13). This cross-reactivity works both ways, ie antibodies to ZIKA can enhance Dengue virus infection in animal models (14), which could be a genuine problem for a prospective ZIKA vaccine. So far though, this issue seems to have been largely ignored.
Indeed, Pardi et al. (8) did not address this issue in their report. These authors developed a mRNA vaccine coding for the MHC class II signal peptide as well as the ZIKA pre-membrane and envelope glycoproteins (preM-E). These proteins are highly immunogenic in other flaviruses and when expressed together result in secretion of subviral particles. The mRNA was in vitro-transcribed using 1-methylpseudouridine instead of uridine, capped with a cap1 and encapsulated in lipid nanoparticles (LNP) for delivery. A single intradermal injection of 30 µg ZIKA prM-E mRNA-LNP in two different mouse strains (C57BL/6 and BALB/c) resulted in protein E-specific IgG levels of ~200,000 from week 2. Neutralizing antibody reached a maximum of 1,100-1,300 at week 8 and persisted up to week 20 as measured via a plaque-reduction neutralization assay. Immunized mice were completely protected against viremia after challenge with 200 plaque forming units (PFU) of a different ZIKA strain 2 or 20 weeks later.
In macaques, a single intradermal injection of 50, 200 or 600 µg also induced high IgG levels, with a maximum of 300,000 at 4 weeks with only a slight drop to 100,000 at week 12, while neutralizing antibody levels reached 400. Again, animals were protected against viremia after challenge with 1,000 TCID50 (50% Tissue culture Infective Dose) of ZIKA at week 5. These levels are 50 times higher than what has been achieved with a 1 mg dose of a ZIKA-DNA vaccine. (15) Interestingly, there was no statistical difference between the dose groups, suggesting that the lowest dose was sufficient to induce maximal immune response.
In contrast, Richner et al. (9) consider the potential for unwanted ZIKA vaccine ADE of Dengue virus infection in their study. In addition to ZIKA prM-E mRNA constructs containing either the signal sequence from human IgE or Japanese encephalitis virus (JEV), they also investigated constructs where amino acids in or near the conserved fusion loop (FL) in the DII domain of the E protein were mutated to abolish FL-specific reactivity. This epitope is immunodominant in humans and likely to be the main driver of ADE between ZIKA and Dengue viruses.
As in Pardi et al., mRNA constructs contain 1-methylpseudouridine instead of uridine, but a proprietary nucleoside modification is mentioned as well. Richner et al. also deliver the mRNA vaccines with LNP, but inject one or two doses intra-muscular and use significantly lower doses for their mice studies (2 and 10 µg compared to 30 µg). Vaccination of immunocompromised AG129 mice lacking interferon signalling responses with prime and boost injections of either 2 or 10 µg IgE-prM-E mRNA resulted in neutralizing antibody titers of >1,000 EC50 at 6 weeks, while single injections were less efficient. With one exception in the 2 µg prime/boost group, mice immunized with either the single 10 µg injection or the prime/boost strategy using 2 or 10 µg all survived challenge with 1000 PFU at week 6. In immunocompetent C57BL/6 antibody titers were lower after the first injection, but rose to >10,000 after boosting at 4 weeks and remained high at 18 weeks. Animals were also protected against lethal challenge with 1,000,000 units of ZIKA virus, which killed 70% of control mice.
In BALB/c mice, prime/boost immunization with both the wild-type and fusion loop modified IgE-prM-E vaccine at 2 or 10 µg resulted in neutralization titers of 5,000 while the JEV signal peptide containing wild-type induced significantly stronger responses, with values up to 100,000. The fusion loop modified JEV-prM-E vector was less immunogenic, but still elicited a stronger response than the IgE peptide-containing constructs at 10,000. Only JEV-prM-E vaccinated animals showed no viremia after ZIKA challenge at 13 weeks. The other groups had break-through viremia at 10-100-fold lower levels than control mice, with little to no detectable viral RNA in uterus and brain tissues.
Experiments in K562 cells demonstrated that sera from mice vaccinated with the wild-type IgE and JEV-prM-E constructs did indeed enhance infection with Dengue virus serotype 1 pseudoviral particles while sera from mice treated with fusion loop mutated constructs could only do so at very high concentrations. Transferral of pooled sera from mice immunized with wildtype constructs into AG129 mice and subsequent challenge with a non-lethal dose of Dengue serotype 2 resulted in lethal infection due to ADE. Sera from mice treated with fusion loop mutant vaccines resulted in significantly less morbidity and mortality.
These results suggest that ZIKA virus vaccines containing the wild-type envelope protein could be very detrimental by significantly enhancing the severity of Dengue infection. According to the WHO, several phase I ZIKA vaccine studies employing attenuated or inactivated ZIKA virus which contain E protein are currently underway although some candidates are specifically aimed at avoiding ADE. (16) However, this potential issue only serves to highlight the big advantage of mRNA vaccines: once they are properly validated, developing a modified mRNA vaccine will only be as difficult as synthesising a different DNA template for in vitro transcription. It is clear that mRNA vaccines are highly effective in animal models and preliminary data from an ongoing phase I trial (NCT03076385) of a mRNA vaccine for influenza strain H10N8 in humans are encouraging. All 23 subjects vaccinated with a single intra-muscular injection of 100 µg H10N8 mRNA vaccine had hemagglutination inhibition (HAI) values ≥ 40, a level generally considered to be protective at day 43. (17) This is comparable to levels achieved with live inactivated influenza vaccines. First results from a phase I trial (NCT03014089) of Moderna’s Zika vaccine mRNA-1325 (a company presentation suggests this is based on the Richner (8) paper) are expected later this year.
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