FAPESP is supporting the development of nine COVID-19 vaccines

Butantan Institute produces CoronaVac and is testing two new vaccines. Four projects are under way at the University of São Paulo, and two at startups supported by FAPESP’s PIPE innovation funding program.

By Elton Alisson and Karina Toledo  |  Agência FAPESP – Besides funding allocated to the Phase 3 clinical trial of CoronaVac, the COVID-19 vaccine produced in Brazil by Butantan Institute, FAPESP is funding eight other research projects to develop COVID-19 vaccines in the state of São Paulo, Brazil.

Some formulations are being tested in animals and should begin clinical trials next year. Four projects are under development at the University of São Paulo (USP), two at Butantan Institute, and two at startups supported by the FAPESP Innovative Research in Small Business Program (PIPE). 

At the Heart Institute (InCor) of the University of São Paulo’s Medical School (FM-USP), a team led by Jorge Elias Kalil Filho is developing a DNA vaccine to be delivered by nasal spray. It will combine part of the spike protein used by SARS-CoV-2 to bind to a receptor and infect human cells with T epitopes, viral antigens that are recognized by the immune system. The goal is to induce a response by neutralizing antibodies in parallel with strong cellular immunity, including cytotoxic T CD8+ lymphocytes, which kill infected cells, and T CD4+ lymphocytes, which assist the production of antibodies and the cytotoxic response.

The protein formed by the mixture of these two components will be produced in cells by means of recombinant DNA technology and carried in nanoparticles that adhere to the nasal mucosa to trigger an immune response and prevent the virus from spreading throughout the respiratory tract.

In contact with the nasal mucosa, the new protein will induce production of large amounts of specific antibodies, such as secretory immunoglobulin A (IgA), and will stimulate T cells in the respiratory tract.

According to Kalil Filho, principal investigator for the project “Mapping SARS-CoV-2 epitopes to T lymphocytes and spike protein receptor to B lymphocytes”, the advantages of the new vaccine compared with first-generation vaccines include the fact that it has specific molecular targets and will therefore induce a stronger immune response, and that it is stable at room temperature.

“We don’t yet know whether the vaccines available now can prevent nasal infection, so we set out to develop a formulation that can be administered nasally to strengthen the mucosa in the respiratory system and block proliferation of the virus,” Kalil Filho told Agência FAPESP.

To define molecular targets for the vaccine, the researchers studied the immune responses of 200 people who fell sick after being infected by SARS-CoV-2. The analysis served as the basis for development of a vaccine with varying compositions, which they tested in mice to define the best antigen, formulation, and nanoparticle. “We now have what can be called a prototype vaccine,” Kalil Filho said.

The researchers are currently working on the production of a cell line that can be used to produce the novel protein on a large scale for industrialization. They expect to begin testing in humans in 2022.

The vaccine can be administered as a booster since most of the population will have been immunized by the time it is ready. “It can be used to induce a very strong local immune response while also reinforcing immune memory,” Kalil Filho said.

Another idea is to combine it with a bivalent intranasal vaccine using the influenza virus to express the SARS-CoV-2 spike protein. This strategy is being followed by researchers at the University of São Paulo’s Ribeirão Preto Medical School (FMRP-USP) under the leadership of Ricardo Tostes Gazzinelli.  

“The benefit of combining the two vaccines would be the production of an augmented immune response,” Kalil Filho said.

Reverse genetics

Gazzinelli is responsible for the project “Bivalent intranasal vaccine using influenza virus to express SARS-CoV-2 spike protein: protection mechanisms and lung injury”. 

The challenge is to develop a DNA vaccine based on influenza reverse genetics in which a gene required by influenza virus to exit the host cell is replaced with a gene that encodes a segment of the spike protein known as the receptor-binding domain (RDB).

The nonreplicating virus produced in this manner will infect nasal mucosa cells and express the SARS-CoV-2 spike protein as well as proteins proper to influenza virus. It will be unable to leave the cells and cause disease, but will nevertheless induce an immune response.

First, the researchers used reverse genetics to produce RBD-expressing influenza virus and administered it nasally to mice. Immunogenicity testing showed that the virus-induced production of anti-RBD antibodies in bronchoalveolar lavage (BAL) and serum from the inoculated animals, as well as a strong response by specific T lymphocytes.

The induced production of antibodies is not yet satisfactory, however. “We’re trying to improve the formulation so that the vaccine induces a higher level of antibody production,” Gazzinelli told Agência FAPESP.

After completing this stage, the researchers plan to perform more tests on immunized animals that will be challenged with SARS-CoV-2, and to begin clinical trials in 2022.

Besides nasal administration of the vaccine, they have also tested intramuscular injection.

According to Gazzinelli, the reverse genetics technique utilized permits a change of hemagglutinin, the main target for antibodies against seasonal influenza virus, so that the vaccine can be bivalent, conferring protection against both COVID-19 and seasonal flu.

Another advantage, in his view, is avoidance of homologous prime-boost, a problem characterized by decreased effectiveness of the vaccine’s second dose, as normally occurs with vaccines that use adenovirus vectors owing to the development of antibodies against the viral vector.

“In the case of adenovirus vaccines against COVID-19, such as Sputnik V, the problem is surmounted by using a different adenovirus in the second dose. In the case of the vaccine we’re developing, the solution is to change the hemagglutinin from one year to the next. The vaccine functions as if it contained two different viruses,” Gazzinelli explained.

Using it as a booster for the vaccine under development by Kalil Filho and his group at InCor would also avoid this problem. “Once both are working, we’ll test them together and separately to find the best strategy for administering them,” Gazzinelli said.

Virus-like particles

A research project led by Gustavo Cabral de Miranda at the University of São Paulo’s Biomedical Sciences Institute (ICB-USP) aims to develop a vaccine using virus-like particles (VLPs).

VLPs have similar characteristics to viral peptides and proteins such as the spike protein used by SARS-CoV-2 to bind to a receptor in human cells and infect them. VLPs are easily recognized by the immune system but contain no viral genetic material and are noninfectious – hence their potential use in vaccines.

To make sure they trigger an immune response, the VLPs are inoculated together with viral antigens (substances that stimulate the production of antibodies). This approach combines the adjuvant properties of VLPs with the targeting of specific antigens. In addition, Cabral explained, VLPs can easily be broken down by the organism because they are natural biological components. 

“Our strategy will enable us to direct the immune system to recognize VLPs conjugated with viral antigens as a threat, and to trigger an immune response effectively and safely,” he said.

The group has developed different vaccine formulations and tested them in animals. One is based on the spike protein’s RDB and so far appears capable of inducing an immune response that neutralizes the virus. “The animal test results suggest this formulation neutralizes the virus with two doses. In the next two months, we’ll improve it to get the same results with a single dose,” Cabral told Agência FAPESP.

The next step will be to develop a cell line that can be used to produce the protein on a large scale. They expect to begin testing in humans in 2022. 

According to Cabral, the plasticity of the technology is an advantage, as it can be used in developing vaccines against other viruses, such as zika, chikungunya or dengue.

Genetic modification of proteins

The other project in progress at ICB-USP, entitled “Development of SAPN nanovaccines against SARS-CoV-2 using S and N as antigens”, is part of the postdoctoral research of Mariana Favaro, with Luis Carlos de Souza Ferreira as principal investigator.

“Our project concerns genetic modification of viral proteins so that they acquire the capacity to self-assemble in a nanoparticle with a three-dimensional structure that closely resembles the morphology of viruses and can therefore interact more effectively with the immune system,” Favaro said. The strategy mimics characteristics of the virus, such as size and repeating antigen presentation, that are naturally recognized by the immune system as signs of pathogens and activate an immune response.

The researchers had already used the strategy to develop a zika vaccine. “We observed a sharp increase in the antibody-mediated response,” Favaro said. “It’s worth noting that similar strategies, such as VLPs, are used commercially against hepatitis B and HPV. The main difference is that VLPs use sequences that exist in nature with the natural capacity to self-organize, whereas here we’re using synthetic sequences developed for this purpose.”

Vaccines based on self-assembling protein nanoparticles (SAPNs) are an advance mainly on subunit vaccines, which only use proteins specific to the pathogen, he added: “Subunit vaccines are a very safe alternative because they don’t use the complete virus, which facilitates their production, but they can be less effective. This difficulty can be surmounted by using adjuvants and nanoparticles, which offer the additional advantage of not requiring complex production structures or high-safety laboratories. They can be produced in Brazil without major obstacles.”

The project is currently in the preclinical trial stage. “We’ve developed formulations based on different viral proteins, some of which have been characterized and assessed in mice for their capacity to induce an immune response,” Favaro said. “In the case of other formulations, especially some based on the SARS-CoV-2 spike protein, we encountered difficulties with production and we’re now adapting the expression system to enhance the quality of the induced antibodies.”

Platform adaptable to novel variants

Two other projects are under way at Butantan Institute, besides production of CoronaVac. One project is led by Soraia Attie Calil Jorge and is developing a platform to produce VLPs for SARS-CoV-2.

The main advantage of this kind of technology, Jorge explained, is that it avoids having to manipulate the virus, which in the case of SARS-CoV-2 would require a laboratory with a high level of biosafety, available at few research centers in Brazil. Another advantage is that VLPs present the antigen to the immune system more or less as the virus presents it, stimulating both the humoral and cellular immune responses. “In addition, it’s a system that enables VLPs to be modified quickly, which is interesting to deal with variants that are able to circumvent the protection conferred by existing vaccines,” she said.

The group led by Jorge has already “built” the vectors and inserted structural genes for SARS-CoV-2 into the genome of the baculovirus that will transport the genes into cells, where the VLPs will be produced.

“We’re starting to produce VLPs in cells,” Jorge said. “As soon as they’ve been characterized, they’ll be tested in animals. We already produce VLPs for rabies, zika, Mayaro and chikungunya viruses in our lab, and we’re confident that we’ll soon be successful with VLPs for SARS-CoV-2.” The team expects approval for tests in humans to take at least a year.

Inducing an immune response 

The other project under way at Butantan Institute is led by Luciana Cezar de Cerqueira Leite and combines two technologies, based on bacterial outer membrane vesicles (OMVs) – nanoparticles that mimic an infection and efficiently activate the immune system – combined with SARS-CoV-2 proteins. The aim is to induce a broad immune response involving both antibodies and defense cells.

“The quantity of antibodies produced by mice immunized with this vaccine was 100 times larger than normal. They also displayed a cell-mediated response. Both are important for protection against the virus,” Cerqueira Leite said.

The researchers are now at the stage of producing the vaccine. “We have the nanoparticles, and we’re purifying the proteins for coupling,” she explained. “The mice will next be immunized in order to verify neutralizing antibody production capacity and protection. We hope to be ready to start these trials in the second semester. If the results are promising, we will then proceed to production in accordance with best practice and seek approval for clinical trials from ANVISA [Brazil’s National Health Surveillance Agency].”

Cytokine production

The seventh project to develop COVID-19 vaccines is being conducted by Imunotera Soluções Terapêuticas, a USP-spinoff supported by the FAPESP Innovative Research in Small Business Program (PIPE). The principal investigator is Luana Raposo de Melo Moraes Aps, a co-founder of Imunotera. 

The project aims at developing a DNA vaccine that will induce cellular immune responses to SARS-CoV-2 based on the genetics of the Brazilian population. The strategy focuses on designing target sequences that include the viral epitopes most easily recognized by T lymphocytes, which produce cytokines or directly kill infected cells.

The researchers adapted an existing T-cell generating technology used to develop, also with the support of PIPE-FAPESP, a DNA vaccine and purified recombinant protein that activates the immune system – both have proved capable of combating HPV-induced cervical cancer. The immunotherapy drug in the form of a recombinant protein has been tested with good results on patients with precancerous lesions caused by HPV treated at Hospital das Clínicas, the hospital complex run by the University of São Paulo’s Medical School (HC-FM-USP).

To develop a COVID-19 vaccine, they first selected the most frequent human leukocyte antigens (HLAs) in the Brazilian population to identify the epitopes most easily recognized by T lymphocytes, especially the CD8+ type. HLAs are a special group of proteins located on the surface of almost all cells in the body.

“Initially we focused on the Brazilian population because no COVID-19 vaccines were available in Brazil when we submitted the project, but the vaccine can be used anywhere in the world. The HLAs we selected are frequent in the American and other populations, for example,” Aps told Agência FAPESP. One of the advantages of basing the vaccine on the genetic profile of a population is that the immune response is better targeted, increasing the effectiveness of the vaccine, she added. 

The vaccine has been tested on mice, which were given two doses, and their immune response was assessed two weeks later. The results of this proof of concept in terms of immunogenicity were satisfactory. The researchers now plan to perform tests on animals infected with SARS-CoV-2 and to begin clinical trials in 2022.

According to Aps, one of the main advantages of this vaccine compared with first-generation vaccines could be its capacity to neutralize variants of concern, as the relevant mutations in SARS-CoV-2 occur in the spike protein, which is not the vaccine’s target.

“This will have to be confirmed by genetic analysis, but the formulation we’re developing has the potential to transcend the limitations of existing vaccines and combat all variants, inducing a robust cellular response in the Brazilian population,” she said.

The vaccine will be injectable, administered in two doses, and stable at room temperature.

Besides the DNA vaccine against COVID-19, the startup is developing an RNA vaccine that looks highly promising, according to Aps.

Oral vaccine

Another project supported by PIPE-FAPESP is being conducted by Invent Biotecnologia in Ribeirão Preto, with Marcel Montels Trevisani as principal investigator.

The firm uses a vaccine platform based on an attenuated live bacterium, which briefly colonizes the lymphoid organs associated with the intestines, as well as secondary lymphoid organs in animals. “The vaccine’s genetically modified vector contains genes that induce expression of SARS-CoV-2 proteins. If the plan succeeds, this combination will stimulate sufficient humoral and cellular immune responses to prevent the development of COVID-19,” Trevisani said.

Because the vector is a live bacterium, this initial stage of the research depends only on a fermenter to grow bacteria to scale. The methodology is tried and tested. “This platform has been used to prevent horse pneumonia caused by Rhodococcus equi, with patent applications in Brazil and elsewhere,” he explained.

In the case of equine pneumonia, all the tests and protocols used throughout the research project show that the vector colonizes the lymphoid organs associated with the intestines and secondary lymphoid organs long enough for the maturation and selection of clones that are highly effective in controlling infections caused by R. equi.

In addition, the researchers detected robust production of IgA (immunoglobulin A, which protects mucosa) and IgG (immunoglobulin G, important and widely dispersed for opsonization and neutralization of infectious agents), as well as a strong cellular immune response capable of killing infected cells. 

“Based on our prior experience, we expect the same to happen with prevention of SARS-CoV-2,” Trevisani said. “We’re currently working on construction of the vaccine platform and measuring expression. The timetable calls for animal testing to begin in August 2021. However, our experience with the platform for R. equi pointed to strong humoral, mucosal and cellular immune responses. In fact, the scientific community in the field considered it one of the top five vaccine proposals for the disease.

“The main advantage of the technology is its applicability. The vector can be administered orally and doesn’t require needles or trained professionals. The scale of vaccine rollout can be huge compared with intramuscular immunization. Also, the fact that it’s based on a live bacterium means the formulation can be lyophilized and used in remote areas of Brazil and the world without requiring cold chain transportation.”

The team does not yet have plans for clinical trials. “Everything depends on the efficacy and safety demonstrated in the preclinical trials involving animals,” Trevisani said.