Ariel Sobarzo

Profile and Persistence of the Virus-Specific Neutralizing Humoral Immune Response in Human Survivors of Sudan Ebolavirus (Gulu)

To better understand humoral immunity following ebolavirus infection, a serological study of the humoral immune response against the individual viral proteins of Sudan ebolavirus (Gulu) in human survivors was performed. An enzyme-linked immunosorbent assay specific for full-length recombinant viral proteins NP, VP30, VP40, and GP1-649 (GP lacking the transmembrane domain) of Sudan ebolavirus (Gulu) was used as well as a plaque reduction neutralization test. Serum samples from human survivors, which were collected up to 10 years following recovery, were screened and analyzed. Results demonstrate that samples obtained 10 years following infection contain virus-specific antibodies that can neutralize virus. Neutralization correlates well with immunoreactivity against the viral proteins NP, VP30, and GP1-649. Sera from individuals who died or those with no documented infection but immunoreactive to ebolavirus did not neutralize. This work provides insight into the duration, profile of immunoreactivity, and neutralization capacity of the humoral immune response in ebolavirus survivors.

Persistent immune responses after Ebola virus infection.

More than a decade after infection with the Gulu strain of Sudan Ebola virus, persons in Uganda were found to have persistent immune responses.

Profiling the Native Specific Human Humoral Immune Response to Sudan Ebola Virus Strain Gulu by Chemiluminescence Enzyme-Linked Immunosorbent Assay

ABSTRACT Ebolavirus, a member of the family Filoviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP1-294 and GP1-649) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP1-649, followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP1-649, and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future.

Immune Memory to Sudan Virus: Comparison between Two Separate Disease Outbreaks

Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda—Gulu 2000–2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1–649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1–649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.

A Serological Point-of-Care Test for the Detection of IgG Antibodies against Ebola Virus in Human Survivors

Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the hosts immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.

Correspondence of Neutralizing Humoral Immunity and CD4 T Cell Responses in Long Recovered Sudan Virus Survivors

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000–2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.

Marburg virus survivor immune responses are Th1 skewed with limited neutralizing antibody responses.

Until recently, immune responses in filovirus survivors remained poorly understood. Early studies revealed IgM and IgG responses to infection with various filoviruses, but recent outbreaks have greatly expanded our understanding of filovirus immune responses. Immune responses in survivors of Ebola virus (EBOV) and Sudan virus (SUDV) infections have provided the most insight, with T cell responses as well as detailed antibody responses having been characterized. Immune responses to Marburg virus (MARV), however, remain almost entirely uncharacterized. We report that immune responses in MARV survivors share characteristics with EBOV and SUDV infections but have some distinct differences. MARV survivors developed multivariate CD4+ T cell responses but limited CD8+ T cell responses, more in keeping with SUDV survivors than EBOV survivors. In stark contrast to SUDV survivors, rare neutralizing antibody responses in MARV survivors diminished rapidly after the outbreak. These results warrant serious consideration for any vaccine or therapeutic that seeks to be broadly protective, as different filoviruses may require different immune responses to achieve immunity.

The Development and Validation of a Novel Nanobody-based Competitive ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in Cattle

Effective management of foot and mouth disease (FMD) requires diagnostic tests to distinguish between infected and vaccinated animals (DIVA). To address this need, several enzyme-linked immunosorbent assay (ELISA) platforms have been developed, however, these tests vary in their sensitivity and specificity and are very expensive for developing countries. Camelid-derived single-domain antibodies fragments so-called Nanobodies, have demonstrated great efficacy for the development of serological diagnostics. This study describes the development of a novel Nanobody-based FMD 3ABC competitive ELISA, for the serological detection of antibodies against FMD Non-Structural Proteins (NSP) in Uganda cattle herds. This in-house ELISA was validated using more than 600 sera from different Uganda districts, and virus serotype specificities. The evaluation of the performance of the assay demonstrated high diagnostic sensitivity and specificity of 94 % (95 % CI: 88.9–97.2), and 97.67 % (95 % CI: 94.15–99.36) respectively, as well as the capability to detect NSP-specific antibodies against multiple FMD serotype infections. In comparison with the commercial PrioCHECK FMDV NSP-FMD test, there was a strong concordance and high correlation and agreement in the performance of the two tests. This new developed Nanobody based FMD 3ABC competitive ELISA could clearly benefit routine disease diagnosis, the establishment of disease-free zones, and the improvement of FMD management and control in endemically complex environments, such as those found in Africa.

Sudan ebolavirus long recovered survivors produce GP-specific Abs that are of the IgG1 subclass and preferentially bind FcγRI

Ebolavirus is a highly lethal pathogen, causing a severe hemorrhagic disease with a high fatality rate. To better understand immune correlates of protection by virus specific IgG, we investigated the evolution of the Fcγ receptors (FcγRs)-activating capabilities of antiviral IgG in serum samples of long recovered survivors. To this end, longitudinal serum samples from survivors of Sudan ebolavirus (SUDV) infection, studied over years, were examined for the presence of Ebola-GP specific IgG subclasses, and for their binding to FcγRs. We developed a cell-based reporter system to quantitate pathogen-specific antibody binding to FcγRIIIA, FcγRIIA, FcγRIIB and FcγRI. With this system, we demonstrate that anti-GP-specific stimulation of the FcγRI reporter by survivors’ sera was substantially high one year after acute infection, with a slight reduction in activity over a decade post infection. We further demonstrate that GP-specific IgG1 is by far the seroprevalent subclass that retained and even enhanced its presence in the sera, over ten years post infection; the prevalence of other GP-specific IgG subclasses was considerably reduced over time. In accordance, GP-specific FcγRI reporter response and GP-specific total IgG1 subclass correlated in the studied group of Ebola survivors. These observations are important for further informing Ebola vaccine and therapeutic development.