Fiocruz study extends knowledge on immune response to Covid-19. Assessing samples from patients admitted with the disease, including people who live with HIV, the research observed an association between the early production of neutralizing antibodies and less time hospitalized with the disease. 

“Neutralizing antibodies are capable of stopping the Sars-CoV-2 from infecting cells. Our study suggests that individuals with a quick production of such antibodies develop less severe conditions,” states the researcher of the Aids and Molecular Immunology Laboratory of Instituto Oswaldo Cruz (IOC/Fiocruz) and coordinator of the study, Dalziza de Almeida.

The research was led by IOC/Fiocruz, in partnership with the Evandro Chagas National Institute of Infectious Diseases (INI/Fiocruz). At IOC/Fiocruz, the Aids and Molecular Immunology and the Functional Genomics and Bioinformatics Laboratories participated in the investigation. The results were published in the international journal Microbiology Spectrum.

In order to dose the neutralizing antibodies, the study applied an innovative methodology, called pseudovirus-based assay. The technique was standardized from the platform implemented for the study of HIV. Compared to the standard method (the plaque reduction neutralization test, known by the acronym PRNT), the pseudovirus-based assay has quicker and more accurate results.

Furthermore, it poses no risk of infection to the researchers, which enables laboratories to run the experiments with a minimal level of biosafety, called BSL1. As it involves handling Sars-CoV-2, the PRNT must be run in a laboratory with level 3 of biosafety (BSL3), due to the high risk to the health of the professionals.

“This methodology facilitates the development of studies on the immune response, which is important to understand the defense mechanisms against the infectious agents and which may help to define the correlates to vaccine protection with higher reproducibility. Reducing difficulties is one of the goals of innovation”, explains Dalziza.

The correlates to vaccine protection are immune markers which can be measured on the blood as indicators of the protection offered by the vaccine. Neutralizing antibodies are often considered as correlates to vaccine protection.

The project, including the implementation of the methodology, was funded by Programa Inova Fiocruz and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Faperj). It also had the support of the National Institutes of Health, of the United States through the HIV Reagent Program.

Quicker recovery

The study characterized the production of neutralizing antibodies in 103 patients hospitalized with Covid-19 between June 2020 and May 2021, prior to vaccination. 

The analyses indicated that most individuals, 88%, had neutralizing antibodies in their blood when they were admitted, suggesting a robust immune response. On the other hand, 12% showed a level of antibodies lower than the minimum detectable on the exam.

In average, the patients with undetectable antibodies were hospitalized for 27 days as opposed to the 14 days of those with robust immune response. In total, 34 deaths were recorded during the study. Among the patients who died, the average antibody titer was 40% below the level observed among those who recovered from Covid-19.

“Time seems to be very important to the effectiveness of the immune response. Among the patients who died, we observed an increase in the production of antibodies after 14 days hospitalized, but that was not enough to hinder the progression of the disease,” observes Dalziza.

The average titer of neutralizing antibodies was similar among individuals with positive and negative results for HIV. None of the patients presented Aids - when the virus affects the functioning of the immune system as to enable other diseases. Most of them were already treating HIV and some had just recently identified the infection.

“We assessed the presence of antibodies for HIV and Sars-CoV-2 on those patients’ serum and saw that they are capable of maintaining the production of antibodies for the two viruses,” highlights Dalziza, adding that one of the questions during the research was if the coinfection would be associated to more severe levels of Covid-19.

“In our study, we observed that patients with HIV were younger than the other patients admitted with Covid-19. That corroborates the idea that the infection by HIV can cause aging of the immune system, increasing the risk of the disease becoming worse,” adds the researcher.

Platform to assess neutralizing antibodies

Pseudoviruses could be described as hollow viruses, as they have the outer structure of a virus, called an envelope, without the functional viral genetic material inside. Those particles are able to infect cells, but not to replicate, as they do not have the complete genome. That is why they do not pose a risk of infection by the researchers and do not require handling in laboratories with a high level of biosafety, as with the Sars-CoV-2 virus.

Pseudoviruses also carry a gene that is activated after infection, which produces a protein that emits photons when oxidated, thus allowing to detect its presence and quantify the infection with a luminescence reading equipment. 

That feature is an advantage to detect and quantify neutralizing antibodies in laboratory assays. The more neutralizing antibodies in the patient’s serum, the fewer particles of the pseudovirus are able to infect the culture of a cell, which can be measured through the level of luminescence emitted. “The assay with pseudovirus is very reliable to antibody titer and shows results in 48 hours,” highlights Dalziza.

In order to produce pseudoviruses, the researchers use genetic engineering techniques. Certain parts of the viral genetic code are inserted into cells, which work as factories, producing viral proteins and assembling the pseudovirus particles.

That is a complex technique that was introduced to the Aids and Molecular Immunology Laboratory of the IOC/Fiocruz for studies on HIV since 2012. The project was coordinated by researcher Vera Bongertz, biochemist who joined the team responsible for the first isolation of the HIV virus in Brazil, in 1987.

In order to adapt the methodology to Sars-CoV-2, the scientists used the already established platform, building a hybrid particle: a pseudovirus with an envelope similar to the one in HIV, but with the coronavirus Spike protein, known as S protein.

“The S protein is responsible for enabling the access of coronavirus into the cells. With a pseudovirus with that molecule, we can detect antibodies capable of bonding with Sars-CoV-2 and preventing the infection,” explains Dalziza, who had worked in the implementation of the methodology for HIV and standardized the technique for coronavirus at the IOC.

With the technique implemented, other research has been running, including analyses on the production of antibodies triggered by the Covid-19 vaccines and the immune response to coronavirus variants. “This work is the result of Instituto Oswaldo Cruz’s innovation policy and the scientific cooperation,” stated Dalziza, highlighting the partnership with the professionals of the Covid-19 Hospital Center and the study team of Recover-SUS, coordinated by researcher Beatriz Grinszteijn, of INI/Fiocruz. “We are open to new projects and cooperations in research involving the response of neutralizing antibodies,” she added.

Source: Fiocruz News Agency (AFN).