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. 2012 Nov;19(11):1844-52.
doi: 10.1128/CVI.00363-12. Epub 2012 Sep 19.

Profiling the native specific human humoral immune response to Sudan Ebola virus strain Gulu by chemiluminescence enzyme-linked immunosorbent assay

Ariel Sobarzo  1 , Eddie Perelman, Allison Groseth, Olga Dolnik, Stephan Becker, Julius Julian Lutwama, John M Dye, Victoria Yavelsky, Leslie Lobel, Robert S Marks
Affiliations

Profiling the native specific human humoral immune response to Sudan Ebola virus strain Gulu by chemiluminescence enzyme-linked immunosorbent assay

Ariel Sobarzo et al. Clin Vaccine Immunol. 2012 Nov.

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 (GP(1-294) and GP(1-649)) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP(1-649), followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP(1-649), 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.

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Figures

Fig 1
Fig 1
Overview of SUDV-gul recombinant gene construction. All cloned recombinant genes were sequenced and compared to the previously published sequence of SUDV-gul. For each gene, the locations of the Flag tag, along with the positions of nucleotide variations in the cloned genes compared to the published sequence, are indicated. The location of each nucleotide variation in the viral genes is indicated by a star, along with the corresponding nucleotide and amino acid changes. All changes observed in the cloned genes were detected in the viral RNA and reflect variations in viral RNA that was obtained from a different isolate for this study.
Fig 2
Fig 2
(A to F) Western blot analysis results for serum samples using a multichannel protein screening device (Bio-Rad). The expressed recombinant viral proteins of SUDV-gul were blotted individually, or in a mix of two, against a panel containing infected and noninfected samples from the original 2000-2001 outbreak (and validated at that time). A mouse anti-Flag commercial antibody was used as a positive control. All positive immunoreactive serum samples were also screened against a control antigen (lysates of 293T cells expressing pCAGGS without the recombinant proteins of SUDV-gul, for which there was no immunoreactivity in any of the samples [data not shown]). Select serum samples screened on these blots are presented. (A) Screening of four serum samples, of which two were from the noninfected group (lanes 2 and 3) and two from the infected group (lanes 4 and 5) against NP. Positive immunoreactivity is shown in lanes 4 and 5. (B) Screening of four serum samples, of which two were from the noninfected group (lanes 2 and 3) and two from the infected group (lanes 4 and 5), against VP30 and VP40. Positive immunoreactivity against VP40 is shown in lane 4. (C) Screening of three serum samples, of which two were from the infected group (lanes 1 and 2) and one from the noninfected group (lane 3) against VP30 and VP40. Positive immunoreactivity against VP30 is shown in lane 2. (D) Screening of seven serum samples, of which four were from the noninfected group (lanes 2 to 5) and three from the infected group (lanes 6 to 8), against GP1–294 and VP35. Positive immunoreactivity against VP35 is shown in lanes 7 and 8. (E) Screening of four serum samples, of which two were from the noninfected group (lanes 2 and 3) and two from the infected group (lanes 4 and 5) against VP24 and VP40. Positive immunoreactivity against VP24 is shown in lane 5. (F) Screening of four serum samples, of which two were from the noninfected group (lanes 1 and 2) and two from the infected group (lanes 3 and 4), against the viral protein L. No positive immunoreactivity was detected.
Fig 3
Fig 3
(A and B) ELISA immunoreactivity screening results of the SUDV-gul-infected (A) and noninfected (B) human serum sample groups against the different recombinant viral proteins NP, VP30, VP40, GP1–294, and GP1–649 and SUDV complete Ag. Each sample was tested in triplicate and screened against a total lysate of cells expressing a given recombinant viral protein and against expressed mock antigen (total cell lysate not expressing viral protein). The S/N results in the infected serum group (A) demonstrated that the greatest immunoreactivity was directed against the recombinant viral protein NP (33 samples), followed by GP1–649 (27), VP40 (11), GP1–294 (10), and VP30 (7). The immunoreactivity result using an inactivated SUDV complete Ag showed a high number of positive recognitions (40 samples), strongly associated with positive NP recognition. The S/N results in the noninfected serum group (B) demonstrated in total a very low number of positive immunoreactivities, primarily directed against NP (5), GP1–649 (4), and VP40 (4). The result of immunoreactivity against an inactivated SUDV complete Ag using 40 random noninfected samples revealed two positive immunoreactive samples (data not shown).
Fig 4
Fig 4
Schematic representation of the correlation between the recombinant viral proteins NP and VP40 of SUDV-gul positive sample recognition in Western blots (WB) and ELISAs. Data analysis of positive immunoreactivity between assays showed that for NP, a total of 21 samples were detected by Western blotting and 33 by ELISA. Out of these, 15 samples were positive in both assays, 6 only by Western blotting, and 18 only by ELISA. VP40 results demonstrated that in total, 6 samples were detected by Western blotting and 11 samples by the ELISA. Out of these, four samples were detected in both assays, two only by Western blotting and seven only by ELISA. Comparison of positive immunoreactivity between VP40 and NP in each assay demonstrated a correlation of 80% (5 out of 6 samples of VP40 were positive for NP) in the Western blot assay and 81% (8 out of 11 of VP40 were positive for NP) in the ELISA.
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