G Specific Antibody Level against Ebola Viral Glycoprotein and Nucleoprotein in Ebola Virus Disease Survivors and Their Relatives

Ebola virus disease is a complex zoonosis that is highly virulent in humans. Despite its sorely pathogenic and lethal nature, survivors of this infection and even asymptomatic cases are able to develop both humoral and cellular immunity against several Ebola virus (EBOV) proteins. We aimed at determining immunoglobulin G (IgG) antibodies level against two Ebola viral antigens, [IQR: −4.01 to −1.918] for anti-EBOV NP IgG. We observed that IgG levels in survivors were higher than in relatives with a significant difference of about 0.0001. The median value of anti-EBOV IgG level among seropositive relatives was 0.7043 U/ml [IQR: 0.5686 to 3.716] for anti-EBOV GP IgG and 4.05 U/ml [IQR: 0.2765 to 7.759] for anti-EBOV NP IgG respectively. Interes-tingly, we observed that 3.30% of Loreto clinic survivors did not developed anti-EBOV NP IgG antibodies; also about 10% survivors of the SLAESB were not reactive to anti-EBOV NP IgG and 1.43% of these survivors did not express antibodies against the Ebola viral glycoprotein. Our work is consistent with previous published studies showing heterogeneity in both survivors and asymptomatic cases of Ebola infection developing adaptive immunity against EBOV proteins.


Introduction
Antibodies play a crucial role in host defense against viruses, both by preventing infection and by controlling viral replication [1]. It is reported that antibodies also exert their antiviral effects by crystallizable fragment (Fc)-mediated effector mechanisms alongside their capacity to neutralize viruses [2] [3]. This involves a bridge between innate and adaptive immune systems, wherein antibodies form immune complexes that drive numerous innate immune effector functions, including antibody-dependent cellular cytotoxicity, antibody-dependent comple-Journal of Biosciences and Medicines ment-mediated lysis, and antibody-dependent phagocytosis [4]. Several mechanisms modulate antibody-mediated effector functions against virally infected cells and can either protect viral replication or enhance infected cell clearance [5]. This phenomenon has been described in Ebola virus disease infection where the ability of antibodies to provide protection from a lethal Ebola virus (EBOV) challenge has been demonstrated in the context of pre-and post-exposure administration of EBOV glycoprotein (GP) specific monoclonal antibodies (mAbs) [6]. It is reported that Ebola virus disease (EVD) survivors develop both humoral and cellular immunity against several EBOV proteins, including GP, secreted GP (sGP), nucleoprotein (NP) and matrix protein VP40 [7].
Ebola virus genome is mainly constituted of seven genes. The viral RNA contains information about eight proteins: VP24, VP30, VP35, VP40, L, NP, sGP and GP1/2. Each of the protein expressed is known for its multi-functionality, essentially VP35 and GP that present multi-functionality in the pathogenesis process and in the inhibition of immune responses in the host [8]. The only virally expressed protein on the virion surface of EBOV is the glycoprotein (GP) that is critical for attachment to host cells and catalysis of membrane fusion [9]. Ebola virus matrix protein or nucleoprotein (NP) is a key component of the viral ribonucleoprotein complex and the most abundant. It has a distinct function in the replication cycle where it plays critical roles in protecting viral RNA from degradation and in mediating genome encapsidation during virus assembly. At present, all research has focused on these primary activities of NP, and any secondary roles remain to be determined [10].
Studies show that immunological events very early in an Ebola virus infection determine the control of viral replication and recovery or catastrophic illness and death [11]. Recovery from infection is related to orderly and well regulated humoral and cellular immune responses, characterized by the early appearance of immunoglobulin M (IgM) and immunoglobulin G (IgG), followed by activation of cytotoxic cells at the time of antigen clearance from blood. By contrast, fatal outcome is associated with impaired humoral responses and an early activation of T cells unable to control virus replication, followed by considerable intravascular apoptosis [12].
Although many studies have been conducted to understand the role of immunity against Ebola virus disease, the extent of asymptomatic EBOV infection is still unclear. Several studies reported a wide variability of antibodies level among close contacts of infected person and even survivors [11] [13] [14]. This high variability may be ascribed to the different antigens targeted. Thus, we aimed at determining immunoglobulin G (IgG) antibodies level against two Ebola viral antigens (Glycoprotein and Nucleoprotein) in Ebola survivors and household contacts or relatives. Overall, we enrolled 199 participants in two different sites described as follow: -The first set was made of 91 Ebola survivors follow up at the Loreto clinic. All the 91 survivors were tested for anti-EBOV NP IgG antibodies and 41 of them were tested for both anti-EBOV NP and anti-EBOV GP IgG antibodies. It should be noted that these activities were carried out in an emergency context where reagent and consumable supplies were highly challenging.

Laboratory Testing
Ebola virus RNA detection using real time PCR in survivors of the Loreto clinic Given that, this was the first West Africa Ebola outbreak and our first experience with Ebola Virus Disease (EVD), we thought one way of ensuring our safety was to ascertain if viremia is cleared after onset of symptoms in survivors. To perform ELISA in these two sets of samples, optimal sample dilution was previously determined at 1:500 as described by Mafopa

Statistical Analysis
We collected data and entered them in a customized template in EpiData software version 3.1. Statistical analyses were done using GraphPad Prism 7.04. p < 0.05 was considered statistical significant.
We performed EBOV nucleic acid detection in Loreto clinic survivors and the presence of EBOV nucleic acid was not detected (Table 1). To be sure that participants were indeed EBOV survivors, anti-EBOV NP IgG antibodies screening using quantitative ELISA technique was performed. Surprisingly, 3.30% (03/91) of these survivors did not developed anti-EBOV NP IgG antibodies.

Anti-EBOV IgG Antibody Activity Variation Related to Gender
Survivors of this specific objective were aged 2 to 65 years old with an average of 30 years old. We enrolled 15.38% infants (2 to 12 years), 9.9% adolescents (above 12 to 19 years) and 69.23% adults (above 19 to 65 years) and 5.49% did not inform us. Adults were more representative. We used one-way ANOVA test with p statistical significant at 0.05. Figure

Ebola Survivors and Family Members of the Sierra Leone Association of Ebola Survivors Bombali Branch (SLAESB)
Samples collected with the help of Makeni Ebola survivors association were made of 70 survivors and 38 relatives. The average age of survivors was 28 years and the one of relatives was 22 years.

Anti-EBOV NP IgG Level in Survivors and Their Relatives
Interestingly, we observed a significantly higher anti-EBOV NP IgG antibody   Figure   6). The proportion of asymptomatic cases (in order words seropositive cases) with anti-EBOV GP IgG antibodies among household contacts was 13.16%

Anti-EBOV GP IgG Level in Survivors and Their Contacts
(5/38). We noticed that anti-EBOV GP IgG prevalence in survivors was 98.57% (69/70) with only 1.43% (1/70) of survivors who did not express antibodies against the Ebola viral glycoprotein.

Anti-EBOV NP IgG and Anti-EBOV GP IgG Level in Survivors
There was no significant difference (P = 0.1702, Figure 7)     observe that about 10% (7/70) of these survivors did not express antibodies against the Ebola viral nucleoprotein and 1.43% (1/70) did no present both antibodies against the nucleoprotein and the glycoprotein.

Anti-EBOV NP IgG and Anti-EBOV GP IgG Level in Relatives of Survivors
Family members who took care of survivors from the Ebola association expressed more anti-EBOV NP IgG. We observed in Ebola seropositive asympto-  Figure 8). About 7.89% (3/38) of the asymptomatic cases expressed both anti-EBOV NP IgG and anti-EBOV GP IgG.

Comparison of Anti-EBOV NP IgG and Anti-EBOV GP IgG Level in the Two Sets of Survivors
When comparing the two sets of samples, we observed that anti-EBOV IgG level against the nucleoprotein were elevated in Loreto clinic survivors (median =

Anti-EBOV-IgG Prevalence
Our findings show that anti-EBOV IgG was detected in both Ebola virus disease survivors and their family members, although at different prevalence rates, suggesting that the immunity, previously reported as persistent among survivors, may be important for protection that allowed subclinical disease among household contacts [17]. Anti-EBOV IgG positive proportion among survivors (98.57% for anti-EBOV GP IgG and 90% for anti-EBOV NP IgG) was higher than in asymptomatic household contacts (13.16% for anti-EBOV GP IgG and 21.05% for anti-EBOV NP IgG) with a significant difference of about 0.0001. It means in order words that immunological response, which occur early in survivors, compare to patients with fatal outcome lead to a more robust antibody response since they encountered high viral load than in asymptomatic patients [11]. Our findings are consistent with the study of Colavita et al. 2019 [18] where they demonstrated that in the late phase of infection, survivors showed high level of pro-inflammatory mediators, which plays a role in the immune system. Thus Anti-EBOV IgG production is strongly stimulated to control virus replication and disease progression. The presence of anti-EBOV IgG in plasma sample of some household contacts is indicative that they came across the virus but were able to clear the infection. This could be due to host factors or low viral load in their blood stream given enough time for a robust humoral immune response and subsequent viral clearance. This low viremia could be because in some cases virus infection resulted in the synthesis of viral proteins without the production of progeny virus [19]. In addition, as reported by Richardson

How Long Ebola Virus Could Stay in the Blood Stream of Survivors?
Many studies reported that Ebola virus could persist in body fluids including semen and ocular fluid months after disease onset [21] [22]. We though one way of ensuring our safety was to ascertain the fact that it is stated that plasmatic Ebola virus is cleared two weeks to 21days after onset of symptoms in survivors [ [15] who observed in their study the lack of detectable antibodies level in some survivors (2.3%). They stated that it could also reflect immune defects resulting in low and/or short-lived antibody responses or could be due to technical errors or miscommunication during sample collection.

Anti-EBOV IgG Antibody Activity Variation Related to Age
Although the first suspected case of the 2013-2016 outbreaks is believed to be a Ebola survivors' ages in our study ranged between 2 to 65 years old with an average age of about 30 years old. We grouped them into three major categories: 15.38% children (2 to 12 years), 9.9% adolescents (above 12 to 19 years) and 69.23% adults (above 19 to 65 years). The fact that adults' survivors were more representative confirms they were the one more exposed to the virus in this outbreak. These figures correlate with the above-mentioned findings.
Our findings shown that anti-EBOV IgG level in children (median = 4.876 U/ml, IQR: 1.298 -7.803) was lower than in adolescents (median = 8.417 U/ml, IQR: 8.205 -8.883) and in adults (median = 7.105 U/ml, IQR: 3.917 -8.659) with a significant difference of p = 0.0171 and p = 0.0462 respectively. Children immune system is immature compare to teenagers and adults' immune system and thus most often less active when challenged by a pathogen. These results differed  [36] stated that the high proportion of anti-EBOV GP IgG might be due to unrelated nonspecific binding, with the high degree of glycosylation in EBOV GP lending itself to such nonspecific recognition. They also added that alternately, it might be that GP is more sensitive than other viral proteins to the presence of cross-reactive antibodies directed against related viruses. Thus, anti-EBOV GP IgG alone may not be sufficient as a marker for demonstration of previous exposure, especially in asymptomatically infected or otherwise unrecognized EVD survivors [37].
We noticed that anti-EBOV GP IgG and anti-EBOV NP IgG seroreactive rates The absence of healthy control group constitutes a limitation to our study. It would have been interesting if we were able to differentiate household contacts who took care of survivors from those who only came in contact because they were sharing the same compound with the infected person. In addition, our study may have been enlightened if we did immunoglobulin G detection toward all the Ebola virus proteins. These gaps are mainly because we worked in resources constraint setting during emergency state where reagents and consumables acquisition was a challenge.

Conclusion
Our work, like previous published studies [11]