key: cord-0286552-4fguvm46 authors: Silva, Everidiene K. V. B.; Bomfim, Camila G.; Barbosa, Ana P.; Noda, Paloma; Noronha, Irene L.; Fernandes, Bianca H V.; Machado, Rafael R. G.; Durigon, Edison L.; Catanozi, Sergio; Rodrigues, Letícia G.; Pieroni, Fabiana; Lima, Sérgio G.; Queiroz, Zelita A. J.; Charlie-Silva, Ives; Silveira, Lizandre K. R.; Teodoro, Walcy R.; Capelozzi, Vera L.; Guzzo, Cristiane R.; Fanelli, Camilla title: Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats date: 2021-08-24 journal: bioRxiv DOI: 10.1101/2021.08.24.457520 sha: 641156434b16f4ee8c5b3862f8b8167f19225225 doc_id: 286552 cord_uid: 4fguvm46 The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection. The disease caused by the new coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), known as COVID-19 (Coronavirus infection disease 2019) had it first reports in December 2019, in China, and by the end of January 2020, was defined by the World Health Organization (WHO) as a public health emergency of international importance. COVID-19 outbreak quickly reached virtually all the countries in the world, leading the WHO to change it classification, defining the disease as a pandemic [1] . Less than 2 years after the notification of the first COVID-19 patient, the world records more than 206 million of registered cases of the disease, 4,34 million of deaths caused by this pathology and the emergence of several SARS-CoV-2 variants [2] [3] [4] . The range of clinical presentations of COVID-19 is extremely wide and may vary from an asymptomatic condition, to severe respiratory and multiple organ failure, which may lead to death. The most common clinical symptoms include; dry cough, fatigue, loss of taste and smell, fever and dyspnea, which vary in intensity and may be moderate and comparable to the manifestations of a common cold, or severe, leading to the need for hospitalization and respiratory support due to acute lung injury [5] . In addition to the classic respiratory syndrome, other clinical and laboratory features, such as changes in the activation of blood coagulation pathways, were observed in COVID-19 patients, who may present fibrin deposition and disseminated microthrombosis, including in the nervous central system (NCS) and in the urinary system [6, 7] . The pathophysiological mechanisms that determine the severity of COVID-19 manifestations are still unclear, and may depend on genetic susceptibility, pre-existing conditions; such as diabetes, hypertension and obesity, and individual general health. Main articles and case reports published to date, show the presence of at least two distinct phases in the evolution of COVID-19 pathology; The first, directly triggered by the viral infection, and the second, more severe and generally correlated with a worse prognosis for the patient, promoted by the exacerbated immune response of the host organism, a life-threatening systemic inflammatory syndrome known as "Cytokine Storm" [8, 9] . The SARS-CoV-2 it the seventh member of the coronavirus family able of infecting human beings. In this family, the subtypes SARS-CoV (responsible for the severe respiratory syndrome outbreak in China, in 2003), MERSCoV (from the Middle East respiratory syndrome in 2012) and the new SARS-CoV-2 can cause serious illness in humans, while the subtypes HKU1, NL63, OC43 and 229E are associated with milder presentations [5, 10] . SARS-CoV-2 is a ssRNA-virus, externally protected by a spherical-shaped phospholipid envelope of about 125 nm of diameter, covered by glycosylated Spike proteins (SP), which are the responsible for chemical affinity of the virus to the mammalian cells. SARS-CoV-2 binds the host cell through the interaction between its S protein and the transmembrane isoform of the angiotensin-converting enzyme 2 (ACE2) of host cells, that serves as a receptor, mediating viral entry. The SARS-COV-2 genome consists of approximately 30,000 nucleotides, which encode the structural viral proteins; S, Envelope protein (EP), Membrane protein (MP) and Nucleocapsid protein (NP), associated with protein units of NP, which in turn regulates viral replication replication [11] . SARS-CoV-2 seems to be less lethal than the SARS-CoV or the MERS-CoV, but more infectious, which could be contribute to its pandemic potential [12] . With the rapid spread of COVID-19 throughout the world, the development of vaccines against SARS-CoV-2 became necessary and urgent. Vaccines contribute to the development of immunological memory, thus minimizing the effects of infectious diseases, in a "second" exposition to the pathogen. Pathogen attenuation or inactivation, as well as the production of recombinant bacterial/viral-derived proteins are among the most employed biotechnology strategies for the production of vaccines [13] . Such elements stimulate both cellular and humoral adaptive immune response of the host, triggering the synthesis of specific antibodies against the pathogen, thus preparing it for future infections. Immunization through the vaccines is one of the most effective strategies for the prevention of infectious disease, and have being applied very successfully over the last decades, since it protects not only the patient who receives the immunizing agent, but the whole community, as the immunized person is unlike to become a vector of transmission to other people. The greater the number of people immunized, the lower the chances of a disease develops and becomes pandemic [13, 14] . Based on this assumption, research centers all over the planet, as well as pharmaceutical industries, public and private bodies have been working incessantly on the development of immunizers against SARS-CoV-2 since the beginning of 2020. Currently, a number of different vaccines against COVID-19 are already in emergency use, although the potential of immunizing action of each of these products, as well as the possible side effects that may be caused by them, have not yet been completely clarified. Moreover, in spite of the recent increase in the number of immunized people around the world, some countries are still suffering from the scarcity of available vaccines. Therefore, experimental and pre-clinical studies are still required to provide more details on the physiological mechanisms of immunization, and to contribute to the development of further immunizers against SARS-CoV-2 [13] [14] [15] . Taking the above into consideration, the aim of the present study was to verify the effects of the application of a recombinant protein derived from the viral NP of SARS-COV-2 virus, carried out by our research group through the culture of genetically modified bacteria, in 2 different strains of rats (Rattus norvegicus): Wistar and Lewis, thus verifying the safety of recombinant NP applications and the potential of this protein as an immunizing agent, by evaluating the production of specifics antibodies by sensitized animals. SARS-CoV-2 RNA was isolated from the second Brazilian COVID-19 patient (GenBank: MT 350282.1) [16] , and reverse transcription was performed to obtain the virus Nucleocapsid cDNA, which was used to amplified the nucleocapsid DNA fragment by Polymerase Chain Reaction, using the following primer sequences: All rats had their body weight assessed twice a week and were kept in metabolic cages for 24h, weekly, to measure individual diet (g) and water (mL) consumption. Urine samples were also collected to determine 24h urinary flow (mL/24h) and urinary protein excretion concentration (UPE, mg/24h), using colorimetric methods (SENSIPROT #36 Labtest, Brasil). By the end of the study, after 4 weeks of follow up, rats were subjected to isoflurane inhalation anaesthesia and submitted to a xipho-pubic laparotomy. Blood samples were collected from abdominal aorta for biochemical measurements, while the left kidney, the liver, the brain and the lungs, were fixed in buffered paraformaldehyde, for further histological analysis. Hepatic function was evaluated through biochemical dosage of proteins and enzymes in the serum samples of studied animals. Total serum protein was determined using Biureto Blood was individually drawn from rats into tubes containing anticoagulant and To verify the success of NP sensitization, we analyzed the presence of specific IgG anti-NP in the serum of inoculated animals by Western Immunoblotting (WB The reaction was developed with a Clarity Western Substrate kit (BioRad) and the image was captured in an Alliance Q9 chemiluminescence imaging system (Uvitec). Bands intensity was analyzed using ImageLab software (BioRad). Samples from kidney, liver and lungs were dehydrated and paraffin-embedded through conventional techniques. "Blind" histological analyses of these organs were performed in 4-μm-thick sections, stained with hematoxylin-eosin. Results were presented as Mean ± SE. All analyses were performed using the GraphPad Prism 7® software. Student's t-test statistics was applied to compare the results obtained in groups NP vs. respective Control. Means were considered statistically different when p<0.05. The immunization with recombinant NP was safe and well-tolerated by both Wistar and Lewis lineages of Rattus norvegicus As exposed on Table 1 , the 4 subsequent IM injections of 150 g of recombinant NP were not toxic for Wistar nor Lewis strains of rats, since it was not observed any mortality among the experimental groups, and no one of the classic symptoms of physical suffering frequently observed in experimental animals, such as; reduced food or water intake, reduced mobility, impaired weight gain or breathing difficulties, were noticed in the animals included in the present study. Hepatic and renal function of NP animals were carefully analyzed in order to investigate subclinical toxicity signs. Thus, even though the differences observed among the two strains of rats, the hepatic function panels of both Control and NP animals were compatible with the laboratorial reference ranges for healthy rats [18, 19] . As shown in compared to its strain-matched however, this difference was not statistically significant ( Figure 4X ). An illustrative heat map was presented in Figure 4Z . In order to verify whether the animals which exhibited a more prominent humoral immunological response against NP (with higher anti-NP IgG sera concentrations) would also be those who exhibited more exuberant lung histological alterations, a heat map was presented to correlate the production of anti-NP IgG, and immunohistochemistry in lung tissue stained with HE and immunostaining by CD68+ and CD3+ ( Figure 4Z ). As can be seen, a positive correlation between antibody production and immunohistochemistry in lung tissue stained with HE is observed mainly for Lewis rats with a correlation coefficient (r) of 0. Currently, the global efforts are concentrated on massive COVID-19 immunization, through both the distribution of the present vaccines for as many people all over the world, and through the stimulus to the local production of different immunizing agents in as many countries it is possible [11, 22] . In this context, the present study aimed to As widely known, once a superior organism is exposed to invading microorganisms, such as viruses or bacteria, small parts of the pathogen stimulates the B-cells of the host, which in turn, differentiate into plasmocytes and start producing specific antibodies. In a very simplified way, antibodies are proteins able to recognizing and bind to known invading microorganisms, promoting both its neutralization or the opsonization of its phagocytosis by macrophages and neutrophils of the innate immune system of the host. Therefore, once an animal is sensitized to a particular pathogen, the response of its immune system in a future infection (reinfection with the same pathogen) will be faster and more effective, thus preventing the development of a more severe presentation of the disease [22, 23] . In order to evaluate the efficiency of NP to activate the immune response of inoculated animals, we analyzed the presence of specific anti-SARS-CoV2 IgG antibodies in the sera of NP-injected and non-injected rats, using WB. As expected, only sera from animals of the NP groups (both Wistar and Lewis strains) exhibited positivity for anti-SARS-CoV2 class G antibodies, therefore; it seems correct to state that the inoculation with recombinant NP in this experimental protocol succeeded in promoting the sensitization of the animals, with consequent synthesis of specific antibodies able of recognizing SARS-CoV2. Our results suggest the potential use of the recombinant NP described here, as both an immunizing agent and a useful tool for the production of rapid indirect diagnostic tests for SARS-CoV2, since WB strips containing this immobilized protein could be used to recognize sera from patients that have already been exposed to the virus, or have already had COVID-19 vaccine, and therefore would have specific strand-sensitive IgGs. In accordance with our findings, recent studies of Smits and collaborators comparing the specificity of IgG antibody response against SARS-CoV-2 demonstrated that, most of the COVID-19 patients exhibited specific immune response against NP, while few or none immunoreactivity against SP or MP have been found [25] . It is of note that the lower respiratory tract of mammals is constantly exposed to multiple airborne pathogens, and a prompt and effective immune response against such inhaled invaders is crucial for the survival of species [26, PABST 2010] . In this context, during the evolutionary process, a mucosal secondary lymphoid tissue, embedded in the walls of the large airways, the Bronchus-Associated Lymphoid Tissue (BALT) became part of the immune defense arsenal of many mammals. BALT is constitutive during all life phases in some species, including rats and rabbits, but it is absent in healthy adult mice and humans [Sminia et al., 1989 , Troy D. Randall 2010] . Although more studies with different species, more similar to the human beings, regarding the specific pulmonary immune response are still required, we can suppose that, interestingly, the IM immunization with recombinant NP was able to activate the BALT of the rats, as a response of the systemic immune response triggered by the immunization, reflecting in the overactivity of immune cells observed in the lung of NP rats. Our findings suggest that, in spite of the IM administration, our immunizing agent was able to trigger an immune response specifically in the lungs of the injected rats. This is clinically relevant: the immunomodulation being a primary mechanism underlying COVID-19 supports the prioritization of tolerance as a vaccine strategy. The data presented in this manuscript have several implications for our understanding on the cellular and humoral mechanisms triggered by SARS-CoV-2 immunizing components. Based on our results, we can conclude that the inoculation of recombinant NP protein in Wistar and Lewis rats was safe, since it did not promote changes in the main clinical, biochemical and histological parameters studied here, which could indicate toxicity of this compound. Additionally, NP inoculation successfully promoted the production of specific IgG antibodies against SARS-CoV2, indicating its potential use for massive vaccination immunization, especially because it seems to induce specific pulmonary protection, although additional studies are still needed. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. 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