ORF8 & ORF3b antibodies: Accurate serological markers of early & late SARS-CoV-2 infection
The currently available SARS-CoV-2 antibody tests with their traditional serological approaches just tell us - yes or no, which has resulted in an underestimation of asymptomatic and mild infections. The next-generation serologic tests with a broader landscape of antibody responses to a wide range of viral proteins may help us establish the timing of infections too. This editorial describes a recent study (Nature, 17th Aug 2020) that gives an idea about the future of antibody testing in Covid-19.
Serologic tests or the blood tests that look for antibodies in the blood (SARS-CoV-2 antibody tests) form a major component for the diagnosis of recent and past COVID-19 infection. Serological testing is important for the diagnosis and sero-epidemiology of SARS-CoV-2 virus infection and vaccines. Several serology tests are currently in use that primarily assess Spike (S) and on occasion, nucleocapsid (N) antibodies.
Problem with the current antibody tests:
Most SARS-CoV-2 positive patients develop robust T and B cellular responses, along with S1-specific antibody responses that are high- titre and neutralizing, which has formed the basis of serological diagnosis and vaccine development. However, several studies have shown that a proportion of individuals with RT-PCR confirmed COVID-19, do not develop robust antibody responses. This possibility of low or no antibody responses creates a limitation for the traditional serological approaches leading to an underestimation of asymptomatic and mild infection. It may also jeopardize the success of a potential vaccine that targets S alone. Therefore, what we need is a broader landscape of antibody responses to a wide range of viral proteins in order to better detect the immunogenicity of SARS-CoV-2 infection and understand pathogenesis and immunity.
Let's first understand the SARS-CoV-2 Genome:
SARS-CoV-2 is a single-stranded RNA virus with a genome size of 29,903 nucleotides, which makes it the second-largest known RNA genome. The virus genome consists of two untranslated regions (UTRs) at the 5′ and 3′ ends and 11 open reading frames (ORFs) that encode 27 proteins.
An open reading frame is a portion of a DNA molecule that, when translated into amino acids, contains no stop codons. The first ORF (ORF1/ab) constitutes almost 2/3 of the virus genome and encodes 16 non-structural proteins (NSPs). The remaining 1/3 of the genome encodes 4 structural proteins, and about seven accessory proteins.
- 4 structural proteins= spike glycoprotein (S), matrix protein (M), envelope protein (E), nucleocapsid protein (N).
- 7 accessory proteins= ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, and ORF10
Structural Proteins and the antibodies against them:
- We need to study antibodies to the four structural proteins = S, N, M and E. Also, antibodies to three S subunits of S = S1, S2 and S2′.
- Seroconversion to structural proteins S and N generally occurs in 2- 3 weeks of illness and is not suitable for diagnosis of acute disease and these assays have suboptimal sensitivity and specificity.
- The S protein has multiple stages of maturation, which may present unique antigens. First, the S protein is cleaved into S1 (contains RBD) and S2 subunits. Next, the S2 is further cleaved into S2′ to form the viral fusion peptide.
- Most vaccines in development against COVID-19 target the S protein to elicit neutralizing antibodies to block infection, as the S protein contains the receptor-binding domain, which is critical for viral entry. However, it is unclear whether neutralizing antibodies to S protein are the major contributor to a protective immune response.
Open Reading Frames (ORF)
- ORF1ab = encodes for a large polyprotein that is proteolytically cleaved into 16 nonstructural proteins (NSP 1 to 16).
- Seven other ORFs that are available = ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8 and ORF10. They may encode for proteins but their functions are yet to be determined.
A recent study tested antibodies to the four structural proteins (S, N, M and E), three S subunits (S1, S2 and S2′), the seven available ORFs (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8 and ORF10) and one relevant NSP within ORF1ab (NSP1).
This study reports the detection of antibody responses directed against an extensive spectrum of 15 SARS-CoV-2 antigens to identify new and unique antigenic targets of the humoral immune response of COVID-19 patients.
Significant findings of the study:
1. Antibody responses to structural proteins vs non-structural proteins
The study showed that patients produce antibodies to structural and nonstructural proteins beyond S. N showed elevated antibody responses, better performing as a diagnostic tool than the other structural proteins.
2. Non-structural Protein N and ORF antibodies:
ORF8 antibodies (in addition to N protein) due to their immunodominance and specificity, form a major marker of acute, convalescent and long-term antibody response to SARS-CoV-2. The combined use of ORF3b and ORF8 provides a highly sensitive and specific method for the detection of patients with COVID-19, both in early and late stages of infection. Combinational use of ORF3b, ORF8 and N can be a high-performing marker of infection at early and late time points.
3. Avoiding the confusion with pre-existing cross-reactive antibodies against other human coronaviruses:
Endemic human coronaviruses like the common cold coronaviruses could result in the detection of pre-existing cross-reactive antibodies, which may reduce the specificity of serological assays. However, this study showed an absence of cross-reactivity with N, ORF8 and ORF3b antibodies, which is crucial for their wide use for diagnosis.
4. Accurate diagnosis with ORF3b and ORF8:
ORF3b and ORF8 antibodies are stable over time, highlighting their importance for early rapid diagnosis of patients with COVID-19. The levels of these antibodies were stable up to day 100 post-symptom onset. Highly accurate diagnosis of COVID-19 is possible if two antibody responses- ORF3b and ORF8 are considered together.
Such studies will help antigen targets for vaccine development as well as monoclonal antibody reagents.
 Hachim, A., Kavian, N., Cohen, C.A. et al. ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection. Nat Immunol (2020). https://doi.org/10.1038/s41590-020-0773-7.
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 Yoshimoto, F.K. The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19. Protein J 39, 198–216 (2020). https://doi.org/10.1007/s10930-020-09901-4
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