INTRODUCTION: In the last several years, a new approach to viral vaccine design has shown great promise. Vaccine makers are looking to see if the success can be replicated with other vaccines. Shingles is a viral infection that is caused by reactivation of varicella zoster virus (VZV), causing chickenpox upon primary exposure. The incidence of shingles increases with age due to the decline in natural immunity. In some populations, such as those in Australia, data shows that approximately 50% of the population will develop shingles. Vaccination is the only way to reduce the number of shingles cases. Research has shown that the SHINGRIX vaccine produces a 24-fold increase in T cells, which is 12 times higher than other less effective shingles vaccines. The possible explanation for this boost in long term immunity to shingles was presented in a paper by Cunningham et al1.

DISCUSSION: In October 2017, the FDA approved SHINGRIX (Zoster Vaccine Recombinant, Adjuvanted) for the prevention of herpes zoster (shingles) in adults aged 50 years and older. The previous live attenuated VZV vaccine (Zostavax, Merck Sharpe & Dohme Corp.) showed reduced efficacy as age increases, declining to only 18% in those over 80 years of age. The recombinant glycoprotein E (gE) subunit vaccine, SHINGRIX was developed to overcome these efficacy limitations due to age. This vaccine contains the recombinant VZV gE protein and the AS01B adjuvant system. It is suspected that the use of a single viral glycoprotein with this adjuvant is responsible for the 24-fold increase in CD4 T-cell responses to this vaccine. Cunningham et al1 discusses the details of this adjuvant system. AS01B contains Quillaja sponaria Molina, fraction 21 (QS21; licensed by GSK from Antigenics LLC- Agenus Inc) and 3-O-desacyl-4′-monophosphoryl lipid A (MPL). AS01B stimulates a local and transient activation of the innate response leading to the recruitment and activation of antigen-presenting dendritic cells. QS21 is an adjuvant that induces transient local cytokine responses and activation of dendritic cells and macrophages in muscle and draining lymph nodes in animal models. The toll-like receptor type 3 agonist MPL synergizes with QS-21 to enhance the immune response to the co-administered antigen through the production of interferon-gamma (IFN-γ). Phase I and II trials demonstrated that a single vaccine dose elicits substantial humoral and cell mediated immune (CMI) responses that further increase after a second dose two months after the initial dose. Each vaccine dose combines 50 µg purified gE with the AS01B adjuvant containing MPL (50 µg), QS-21 (50 µg) within liposomes. Overall, vaccine recipients ≥50 years of age showed a 24.6-fold increase in gE-specific CD4 cells. This is in comparison to placebo recipients (saline only) where there was no change in gE-specific CD4 T-cell frequencies after vaccination at any time point. Humoral responses were elevated in all age groups throughout the 36-month observation, with slightly lower immune response in those over age 70 years. While a high, persistent circulating antibody response is induced, gE-specific CMI is believed to be the main mechanistic driver of protection against herpes zoster. The phase II clinical trials induced a gE-specific CMI response in > 90% of recipients. Peak CD4 T-cell frequencies were observed at 1 month following dose 2, then declined substantially by 12 months after dose 2, and remained stable for the remainder of the study. This vaccine has a multi-fold increase in humoral and CMI responses compared to the previous live-attenuated vaccine. It is thought that the ability of SHINGRIX to elicit this substantial immune response in older age groups is likely due to the capacity of AS01B Adjuvant System to enhance gE-antigen presentation by increasing the number of activated antigen-presenting cells. In addition, AS01 promotes T-cell responses through a synergistic effect between MPL and QS-21, involving the stimulation of macrophages in the draining lymph node and early IFN-γ production, which in turn mediates the effects on dendritic cells. Cunningham suggests another factor possibly enhancing the immune response is that the previous live-attenuated vaccine induces a broad response against multiple antigens, while the SHINGRIX vaccine immune response is directed against a single immunodominant antigen, which indicates that a strong narrowly focused immune response can be highly protective, even against a complex viral pathogen that possesses multiple immune evasion pathways.

DISCUSSION: Two doses of the SHINGRIX vaccine induced robust humoral and cellular immune responses in all age groups (especially people ≥70 years). It is thought that the ability of this vaccine to induce such persistent antibody and polyfunctional CD4 T-cell responses in older adults is due to using a single viral antigen in combination with the use of the AS01B adjuvant system which apparently can overcome the immunosenescence seen with age.

  1. REFERENCES:
    1) Anthony L. Cunningham, T.C. Heineman, H. Lal, O. Godeaux, R. Chlibek, S.-J. Hwang, J. E. McElhaney, T. Vesikari, C. Andrews, W. S. Choi, M. Esen, H. Ikematsu, M. K. Choma, K. Pauksens, S. Ravault, B. Saluan, T. T. Schwarz, J. Smetana, C. V. Abeele, P. Van-den-Steen, I. Vastiau, L. Y. Weckx, and M. Levin. (2018). Immune Responses to a Recombinant Glycoprotein E Herpes Zoster Vaccine in Adults Aged 50 Years or Older. JID 2018:217;1750-1760. https://DOI.org/10.1093/infdis/jiy095

INTRODUCTION: In this follow-up report, we discuss several adjuvanted SARS-CoV-2 subunit vaccines in development. In general, subunit vaccines exhibit low immunogenicity and require assistance from an adjuvant to enhance a robust vaccine-induced immune response. The S glycoprotein of SARS-CoV-2 is the preferred viral protein to induce neutralizing antibodies due to its known immunogenic properties. The S glycoprotein possesses two conformational states—a pre-fusion and a post-fusion state. The vaccine antigen must preserve the epitopes in these two conformational states to elicit a good antibody response. There are several SARS-CoV-2 vaccine trials in various clinical Phases, which are using either the GSK adjuvant AS03 (Sanofi & GSK), CpG 1018 from Dynavax (Novavax), or using both adjuvants (Clover Biopharmaceuticals trial).

DISCUSSION:  Liang et al.1 presented results from using both adjuvants. The trimeric spike protein (S) of SARS-CoV-2 binds to ACE2 (angiotensin-converting enzyme)—the host cell surface receptor—and mediates the viral entry via membrane fusion. Based on the recent history of developing vaccines against coronaviruses (such as SARS in 2003 and MERS in 2012), there are at least two challenges in producing safe and effective vaccines against these RNA viruses. The first challenge is inducing broad neutralizing antibodies. The second challenge is reducing or eliminating the possibility of vaccine-associated enhanced respiratory disease, as seen in the case of Respiratory Syncytial Virus (RSV) and SARS vaccines. Liang et al.1 described using a Trimer-Tag platform with a tailored subunit antigen (S glycoprotein) derived from the wild-type viral sequences. The S glycoprotein was subcloned into the pTRIMER mammalian expression vector to allow in-frame fusion to Trimer-Tag, capable of self-trimerization via disulfide bonds. The column purified the S-Trimer protein, which was secreted by CHO cells in a serum-free culture medium. The column bound S-Trimer was purified to near homogeneity in a single step.

Mice immunized with the S-Trimer and an adjuvant of either ASO3 or CpG 1018 induced high levels of neutralizing antibodies. They further studied the immunogenicity of the adjuvanted S-Trimer in nonhuman primates (rhesus macaques). High levels of binding and neutralizing antibody titers were seen, including a boost-effect after the 2nd dose on Day 21. Upon challenge (Day 35) with the SARS-CoV-2 virus, animals were protected from body weight loss and body temperature increases. Complete reduction of viral loads was seen in necropsy lung tissue in both ASO3 and CpG 1018 (plus alum) adjuvanted S-Trimer groups. In contrast, viral loads were detectable in the non-vaccinated (viral challenged) group. This study showed significant adjuvant effects occur with both AS03 and CpG 1018 plus alum with the robust high-level induction of humoral and cell-mediated immune responses to S-Trimer in rodents and nonhuman primates.

Interestingly, Liang et al.1 noted some differences in the immune responses stimulated by these two adjuvant systems. In nonhuman primates, AS03 appeared to induce a stronger humoral immune response, inducing higher levels of neutralizing antibody titers than CpG 1018 plus alum. However, there were no apparent differences in the immune protection against the SARS-CoV-2 challenge observed between the two adjuvant systems in nonhuman primates. The critically important finding was no observation regarding disease enhancement signs, a theoretical concern based on prior experience with vaccine candidates against SARS-CoV-2 and RSV that utilized inactivated viruses.

CONCLUSION: Clinical trials with the SARS-CoV-2 S protein adjuvanted with either AS03 or CpG 1018 plus alum can induce robust humoral and cellular immune responses in various animal species giving protective immunity against SARS-CoV-2 infection in nonhuman primates, with no signs of disease enhancement. This last point is significant since current vaccines using either mRNA, DNA-vector, or inactivated virus vaccines have either done limited animal trials—or bypassed them altogether—to rush them into the market. As these subunit vaccines work their way through clinical trials—Novavax just entered a Phase 3 trial with CpG 1018 adjuvanted S-protein in late December 2020—we will provide updates.

  1. REFERENCES:
    1. Liang, J. G., Su, D., Song, T.-Z., Zeng, Y., Huang, W., Wu, J., Xu, R., Luo, P., Yang, X., Zhang, X., Luo, S., Liang, Y., Li, X., Huang, J., Wang, Q., Huang, X., Xu, Q., Luo, M., Huang, A., Luo, D., Zhao, C., Yang, F., Han, J.-B., Zheng, Y.-T., & Liang, P. (2020). S-Trimer, a COVID-19 subunit vaccine candidate, induces protective immunity in nonhuman primates. bioRxiv. https://doi.org/10.1101/2020.09.24.311027

MKTG 1053 Rev A – 010821

INTRODUCTION: Over the last several decades, subunit vaccines have shown consideration for vaccines. About 30 years ago, scientists developed a potential subunit vaccine for the varicella-zoster virus (VZV).1 A subunit vaccine’s advantage is several-fold, including not needing to grow and inactivate live viruses (or create an attenuated virus strain) and use the most antigenic portion of the virus antigen in question.

 

DISCUSSION: The key to this vaccine is that, unlike the live attenuated VZV vaccine, latency cannot be established when only a part of the virus protein (antigen) is used as the vaccine. In the subunit VZV vaccine’s initial stages, the VZV glycoprotein E (gE ) expresses in a recombinant vaccinia virus vector. Since regulatory agencies did not approve recombinant vaccinia vectors, scientists needed a different path. They constructed secretory truncated VZV glycoprotein gE with 511 amino acids into a vaccinia virus vector which would secret the glycoprotein. Next, they purified this from tissue culture fluids of the infected cells. The secreted gE protein-induced complement-dependent neutralizing antibodies in rabbits. Subsequent research showed that a VZV seropositive individual, when immunized with purified VZV gE in combination with an adjuvant, produced neutralizing antibodies.2  In the subsequent 20 years, GlaxoSmithKline’s clinical phase trials showed promising results with the truncated gE protein. As a result, the US Food and Drug Administration approved the VZV subunit vaccine (which used a GSK proprietary Adjuvant System, ASO1B), now called SHINGRIX, on Oct 20, 2017. Tavares-Da- Silva et al.3 found that based on the sale data from February 2019, with 9.4 million doses of vaccine, the efficacy (>90%) and the vaccine’s safety continue to mirror the clinical trials’ results. With the success of SHINGRIX, others have been encouraged to develop viral subunit vaccines. For example, respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection in infants. Ensuing, scientists developed a recombinant RSV fusion glycoprotein (RSV F subunit). Leroux-Roels et al.4 found a single dose of the RSV subunit F vaccine was well-tolerated and enhanced pre-existing neutralizing antibodies through six months of follow-up. Awasthi, Hook, Shaw, and Friedman5 explain the development of a trivalent herpes simplex virus type 2 (HSV-2) vaccine using HSV-2 glycoproteins C, D, and E (gC2, gD2, gE2) using an alum adjuvant. This trivalent vaccine reduced the frequency of recurrent genital lesions and vaginal shedding of HSV-2 DNA by 50% (shedding of the replication-competent virus was almost eliminated). This result suggests the trivalent vaccine is a worthy candidate for immunotherapy of genital herpes. Furthermore, Tai et al.6 found that using a subunit vaccine containing the envelope protein of Zika is a strong candidate with high efficiency in preventing Zika virus infections in mice.

CONCLUSION: Subunit vaccines are being designed for many virus infections, based, in part, on the successful design and use of the VZV subunit vaccine, SHINGRIX. Only including the most immunogenic components of the virus antigenic protein in a vaccine offers many advantages, not the least of which is not needing to grow a live virus (whether an attenuated strain or a live virus to be inactivated before vaccination). Additional subunit vaccines will be appearing in the not-too-distant future.

 

REFERENCES:

  1. Vafai, A. (1993). Antigenicity of a candidate varicella-zoster virus glycoprotein subunit vaccine. Vaccine, 11(9) 937-940. https://doi.org/10.1016/0264-410x(93)90382-8
  2. Vafai, A. (1995). Boosting immune response with a candidate varicella-zoster virus glycoprotein subunit vaccine. Vaccine, 13(14), 1336-1338. https://doi.org/10.1016/0264-410x(94)00073-v
  3. Tavares-Da-Silva, F., Co, M. M., Dessart, C., Hervé, C., López-Fauqued, M., Mahaux, O.,  Van Holle, L., & Stemann, J.-U. (2019). Review of the initial post-marketing safety surveillance of the recombinant zoster vaccine. Vaccine, 38(18), 3489-3500. https://doi.org/10.1016/j.vaccine.2019.11.058
  4. Leroux-Roels, G., De Boever, F., Maes, C., Nguyen, T. L.-A., Baker, S., & Gonzalez, A. (2019). Safety and immunogenicity of a respiratory syncytial virus fusion glycoprotein F subunit vaccine in healthy adults: Results of a phase 1, randomized, observer-blind, controlled, dosage-escalation study. Vaccine, 37(20), 2694-2703. https://doi.org/10.1016/j.vaccine.2019.04.011
  5. Awasthi, S., Hook, L. M., Shaw, C. E., & Friedman, H. M. (2017). A trivalent subunit antigen glycoprotein vaccine as immunotherapy for genital herpes in the guinea pig genital infection model. Human Vaccines & Immunotherapeutics, 13(12), 2785-2793. https://doi.org/10.1080/21645515.2017.1323604
  6. Tai, W, Chen, J., Zhao, G., Geng, Q., He, L., Chen, Y., Zhou, Y., Li, F., and Du, L. (2019). Rational design of Zika virus subunit vaccine with enhanced. Journal of Virology, 93(17), e02187-18. https://doi.org/10.1128/JVI.02187-18

MKTG 1052 Rev A – 112420

INTRODUCTION

Herpes zoster (HZ) is caused by reactivation of latent varicella-zoster virus (VZV) within the cranial nerve or dorsal root ganglia after primary infection. There is a lifetime risk, ~10% to 30%, of HZ in adults, but is rare in healthy children. Immunocompromised individuals have an increased risk of other diseases, including HZ. Risk of HZ increases by age and a weakened immune system, especially in poor T-cell immunity. In children with cancer, HZ can lead to severe complications, including severe postherpetic neuralgia, visceral dissemination, acute or progressive outer retinal necrosis, and even death. Limited information is available on HZ in children with cancer. Lin et al.1 performed a nationwide population-based cohort study in Taiwan to estimate the incidence of HZ in children with cancer. The study, conducted on the sizeable data-scale dataset available from the National Health Insurance (NHI) program in Taiwan, explored HZ and cancer’s association.

DISCUSSION

In a study by Lin et al.1 , they identified 4,432 newly diagnosed children with cancer between 2000 and 2007 from the outpatient database. As a non-cancer control group, 17,653 children without cancer were frequency matched by sex and age. The average ages at the entry of the cancer group and non-cancer groups were 8.90 and 8.91 years, respectively. The distributions of age at entry, sex, urbanization level, or residential areas, and the prevalence of atopic dermatitis were similar in the two groups. All children received follow-up until death, HZ event, withdrawal from NHI, or end of December 2008. Children with cancer had a significantly lower prevalence of allergic rhinitis and bronchial asthma. The study demonstrated a higher incidence of HZ in children with cancer. The incidence rate of HZ in the population of 4,432 children with cancer was 20.7 per 10,000 person-years, and the incidence rate in the population of 17,653 without cancer was 2.4 per 10,000 person-years. The cumulative incidence was significantly higher in the cancer group (p < 0.0001, or 8.6 times higher). HZ may occur more frequently in children with cancer. More than 80% of children with lymphoma or acute leukemia developed HZ within two years of a cancer diagnosis. A study by Feldman, Hughes, and Kim 2 reported that the overall incidence of HZ in 1,132 children with cancer was 8.9%, and the incidence was 22% higher in patients with Hodgkin disease. Menon and Wan Maziah 3 identified a diagnosis of HZ in 5% (10/188) of the children with cancer and noted the most common malignancy in their study was leukemia.

CONCLUSION

Lin et al.1  found the incidence rates of HZ was 8.6-fold higher in children with cancer than those without cancer. They found that children with cancer were associated with an increased risk of HZ, with those who had leukemia having the highest magnitude of strength association. Early antiviral therapy is mandatory for immunocompromised patients. They concluded that vaccination with either heat-treated zoster vaccine or adjuvanted subunit vaccine (SHINGRIX) could be an appropriate policy to decrease herpes zoster incidence in children with cancer.

REFERENCES

  1. Lin, H.-C., Chao, Y.-H., Wu, K.-H., Yen, T.-Y., Hsu, Y.-L., Hsieh, T.-H., Wei, H.-M., Wu, J.-L., Muo, C.-H., Hwang, K.-P., Peng, C.-T., Lin, C.-C., & Li, T.-C. (2016). Increased risk of herpes zoster in children with cancer: A nationwide population-based cohort study. Medicine, 95(30), e4037. https://doi.org/10.1097/md.0000000000004037
  2. Feldman, S., Hughes, W. T., & Kim, H. Y. (1973). Herpes zoster in children with cancer. American Journal of Diseases of Children, 126(2), 178-184. https://doi.org/10.1001/archpedi.1973.02110190156009
  3. Menon, B. S., Wan Maziah, W. M. (2001). Herpes zoster in children with cancer. The Malaysian Journal of Pathology, 23(1), 47-48. Retrieved from https://europepmc.org/article/med/16329548

By David Kilpatrick, PhD and Abbas Vafai, PhD

MKTG 1051 Rev A – 110220

INTRODUCTION:
In the first nine months of 2020, it is becoming increasingly clear that being infected with the severe acute respiratory syndrome Coronavirus 2 (SARSCoV-2), now commonly referred to as COVID-19, may lead to the reactivation of varicella-zoster virus (VZV), causing herpes zoster (HZ). Read more

INTRODUCTION:
Surprisingly, much of the general public do not know one of the human Herpesviruses, varicellazoster virus (VZV), causes chickenpox, and results in a life-long infection. VZV can re-activate after decades of being dormant in sensory nerve ganglia (usually in those over 60 years of age) to cause shingles infection. Read more

INTRODUCTION:
Herpes zoster (HZ) reactivation is characterized as a vascular rash of unilateral distribution that can also cause complications such as post-herpetic neuralgia, ophthalmic zoster, and other neurological diseases. Emerging epidemiological and clinical data recognizes an association between HZ and subsequent acute strokes and myocardial infarction (MI). Read more

INTRODUCTION:
Patients have a higher risk of HZ with diseases such as rheumatoid arthritis (RA), psoriasis (PsO), and inflammatory bowel-related diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease (CD). Using immunosuppressive therapy, which treats these diseases, increases the risk of HZ.

DISCUSSION:
The most common risk factor for HZ is increasing age, presumably due to a weakening immune system as we age. In approximately 15% of the general population, varicella-zoster virus (VZV) reactivates after a latency period to cause HZ (shingles). Patients with autoimmune diseases, such as RA, IBD, UC, PsO, and CD diseases, have an increased risk of HZ compared to the general population. The risk of HZ increases by the use of immunosuppressive therapy to treat autoimmune diseases. One such drug for treatment is Janus kinase (JAK) inhibition. Tofacitinib, an oral JAK inhibitor for the treatment of RA and psoriatic arthritis, is under investigation for the treatment of UC and previously for PsO. Although there is a dose-dependent risk for HZ when taking tofacitinib, the majority of HZ cases reported are non-complicated, mild to moderate in severity, and manageable with standard antiviral therapy (acyclovir). Vaccination (SHINGRIX) should be considered before treating patients to reduce the risk of HZ patients receiving JAK inhibitors1Colombel, J. F. (2018). Herpes zoster in patients receiving JAK inhibitors for ulcerative colitis: mechanism, epidemiology, management, and prevention. Inflammatory Bowel Diseases, 24(10), 2173-2182. https://doi.org/10.1093/ibd/izy150. Cullen, Baden, and Chiefetz2Cullen, G., Baden, R., P., & Cheifetz, A. S. (2012). Varicella zoster infection in inflammatory bowel disease. Inflammatory Bowel Diseases, 18(12), 2392-2403. https://doi.org/10.1002/ibd.22950 presented a review of publications describing VZV infections in inflammatory bowel disease (IBD) patients. They looked at 20 cases of primary VZV infection with IBD and 32 cases of HZ infections in patients with IBD. Fifteen of the 20 VZV cases had CD, which likely reflects the greater use of immunosuppression in this disease than UC. They identified various immunosuppressive drugs used in 20 patients, including anti-TNF (9 patients), corticosteroids (13), and either thiopurine or methotrexate (12). All 32 cases of HZ in IBD patients were on immunosuppression with corticosteroids, thiopurines, and anti-TNF. Combination therapy increased the risk of HZ even further. However, in a more comprehensive nationwide Veteran Administration study with 295 patients, Khan et al.3Khan, N., Trivedi, C., Shay, Y., Patel, D., Lewis, J., & Yang, Y. (2018). The severity of herpes zoster in inflammatory bowel disease patients treated with anti-TNF agents. Inflammatory Bowel Diseases, 24(6), 1274-1279. https://doi.org/10.1093/ibd/izx115 found that the incidence and severity of HZ in patients on anti-TNF medications were found not to be associated with an increased risk of developing severe HZ among these IBD patients. They believed TNF-α to play an important role in viral clearance, so it was logical to think anti-TNF medications could impair host immune function. Still, the data suggest that IBD patients who develop HZ during anti-TNF therapy are not at increased risk of developing complications from the HZ infection.

CONCLUSION:
Despite the risk of a reactivating HZ infection in persons with autoimmune diseases, such as those described, there are several immunosuppressive drugs available to treat these diseases, while not increasing the further risk of HZ infections. Both tofacitinib and anti-TNF therapies, as referenced above, are two such drugs. There is growing support for patients with IBD to receive vaccination against HZ using the newly released vaccine, SHINGRIX, before immunosuppressive therapy treatment. SHINGRIX vaccination is recommended even if patients have received the previous live virus vaccine.

By David Kilpatrick, PhD and Abbas Vafai, PhD

MKTG 1047 Rev A

INTRODUCTION:
Oral fluids have been used to detect Herpes virus antibodies, including secretory IgA, IgM, and IgG. Herpes virus particles have also been identified in saliva. Several Herpes viruses, such as Epstein–Barr virus (EBV), varicella-zoster virus (VZV), and herpes-simplex-1 (HSV-1), have even been detected in the saliva of Astronauts from shuttle-flights and ISS missions1Cohrs, R. J., Mehta, S. K., Schmid, D. S., Gilden, D. H., & Pierson, D. L. (2008). Asymptomatic reactivation and shed of infectious varicella zoster virus in astronauts. Journal of Medical Virology, 80(6), 1116–1122. https://doi.org/10.1002/jmv.21173 2Rooney, B. V., Crucian, B. E., Pierson, D. L., Laundenslager, M. L., & Mehta, S. K. (2019). Herpes virus reactivation in astronauts during spaceflight and its application on earth. Frontiers in Microbiology. 10, 16. https://doi.org/10.3389/fmicb.2019.00016. The ease of sample collection, along with the cost-effective use of lateral flow assays for detection, opens a wide range of opportunities for easily detecting Herpes viruses in point-of-care settings.

DISCUSSION:
A recent review by Miočević et al.3Miočević, O., Cole, C. R., Laughlin, M. J., Buck, R. L., Slowey, P. D., & Shirtcliff, E. A. (2017). Quantitative lateral flow assays for salivary biomarker assessment: A review. Frontiers in Public Health, 5, 133. https://doi.org/10.3389/fpubh.2017.00133 discusses the strengths and weaknesses of using lateral flow assays (LFAs) for detecting viruses in saliva. The collection of saliva allows for a repeated collection, if needed, without the stress of drawing blood. Even with the advent of LFAs for diagnostic assays in recent years, there are relatively few such assays for viral detection in saliva. LFAs can work either as an immunoassay (LFIA) to detect viral-specific antibodies in the collected sample or to directly detect the virus particle present in the sample. The assay works based on liquid movement (containing the analyte to be detected) across a strip of polymeric material containing dry reagents that activate by the lateral movement of a liquid sample up the strip membrane. The specific detection area on the strip can contain either (1) viral-specific recombinant proteins, to which the viral antibodies in the saliva will recognize by binding to the recombinant viral protein; or (2) viral-specific antibodies on the test strip, to which the virus particle in the saliva will be recognized and bound. Despite the simplicity of this assay description, extensive development of these assays is required by the manufacturer to overcome assay limitations, such as lower analyte concentrations in the sample. Developers are utilizing various approaches such as using colloidal gold or carbon, fluorescent or luminescent materials, or colored latex beads. As an example, colloidal nanoparticles generate direct signals, whereas the use of other materials may require additional steps to derive analytical results, such as upconverting phosphor technology (UPT). UPT is based on sub-micron sized ceramic particles coated with lanthanides that absorb infrared light (excitation) and emit visible light (response signal). The particles functionalize with antibodies and antigens for use as labels on a lateral flow strip. There can be many steps in the assay development to consider including, sample composition and how the sample will flow along the strip, as well as the concentration of the analyte to be detected in the sample. Manufacturers must ensure that only the molecules of interest bind to the antigens or antibodies coated on the test strip.

CONCLUSION:
The use of lateral flow assays for detecting virus particles or virus-specific antibodies is a promising approach when applied to saliva-based assays. There are many advantages to both of these sample collection and detection assays. Although there are several commercial assays to detect Herpes viral nucleic acid in saliva, at present, there are few if any such assays available for detecting Herpes virus analytes (antibodies or virions) in saliva using an LFA.

By David Kilpatrick, PhD and Abbas Vafai, PhD

 

MKTG 1046 Rev A

INTRODUCTION:
Numerous labs are developing antibody assays to detect COVID-19. There are presently (as of May 1, 2020) four FDA approved assays for detecting IgG/IgM and three for detecting IgG only. Four of these assays use a lateral flow assay (LFA) architecture. All of the assays use the viral S1 glycoprotein as the antibody target. This short note will describe a typical LFA for COVID-19.

DISCUSSION:
A rapid point-of-care assay that detects either/or both IgG/IgM is critical for detecting spread on the infection through the population. A recent infection (<7days) is usually seen with the production of IgM, while older infections (>8 days since infection) detect the generation of IgG. Li et al. (2020) developed an assay that detects both IgG and IgM, detecting antibodies to the SARS-CoV-2 S1 spike protein. They purified the recombinant S1 antigen (MK201027) by protein A affinity chromatography and size-exclusion chromatography. They based the design of the S1 antigen on the published SARS-CoV-2 sequence (MK201027). Antibodies obtained from Sigma include bovine serum albumin (BSA), goat anti-human IgG and IgM antibodies, rabbit IgG, and goat anti-rabbit IgG antibodies. Shanghai KinBio Inc. provided 40Nm gold nanoparticle (AuNP) colloids, NC membrane, and plastic pad, and Whatman provided the glass fiber conjugate (GFC). Sigma produced the PBS. Hunan CDC, China supplied inactivated COVID-19 serum and negative serum samples of patients.

To prepare the AuNP conjugate, they added SARS-CoV-2 recombinant protein dissolved in PBS (1mg/ml) to the mixture of 1ml AuNP colloid (40nm in diameter, OD=1) and 0.1ml of borate buffer (0.1M, pH 8.5). After incubation for 30 minutes at room temperature, the mixture was centrifuged at 10,000 rpm at 4oC for 20 minutes. Next, and 1ml of BSA in PBS was added to the AuNP conjugate to be re-suspended after discarding the supernatant. They repeated the centrifugation and suspension twice, and the final suspension was in PBS. The AuNP-rabbit IgG conjugates were prepared/purified by the same procedure. The main body of the test strip consists of five parts, including plastic backing, sample pad, conjugate pad, absorbent pad, and NC membrane. Each component of the strip is pretreated as follows: the NC membrane was attached to a plastic backing layer for cutting/handling. Researchers immobilized the anti-human-IgM, anti-human-IgG, and anti-rabbit-IgG at test M, G, and control line C. Then, they sprayed conjugate pad with a mixture of AuNP-COVID-19 recombinant antigen conjugate and AuNP-rabbit-IgG. Sample pad was pretreated with BSA (3%, w/v) and Tween-20 (0.5% w/v) before use. To run the assay (at room temperature), researchers pipetted 20 ul whole blood sample (or 10 ul of serum/plasma samples) into the sample port, followed by adding 2-3 drops (70-100ul) of dilution buffer (10mM PBS) to drive capillary action along the strip. The test takes approximately 15 minutes to complete. If only the C line shows red, the sample is negative. Either M or G line or both lines turning red indicates the presence of anti-SARS-CoV-2-IgM or IgG, or both if IgG and IgM are in the specimen.

CONCLUSION:
Of the 397 blood samples (vein blood) from SARS-CoV-2 infected patients, 352 tested positive, for a sensitivity of 88.66%. Twelve of the blood samples from the 128 non-infected patients were positive, for a specificity of 90.63%. Also, 256 out of 397 (64.48%) were positive for both IgG and IgM. Patient finger stick blood was tested and showed that all positive/negative results matched with 100% consistency between vein and finger stick blood, indicating the use of this assay as a point-of-care test using fingerstick blood.

By David Kilpatrick, PhD and Abbas Vafai, PhD

Li, Z., Yi, Y., Luo, X., Xiong, N., Liu, Y., Li, S., Sun, R., Wang, Y., Hu, B., Chen, W., Zhang, Y., Wang, J., Huang, B., Lin, Y., Yang, J., Cai, W., Wang, X., Cheng, J., Chen, Z., Sun, K., Pan, W., Zhan, Z., Chen, L., & Zhang, Y. (2020). Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS-CoV-2 infection diagnosis.

Journal of Medical Virology. https://doi.org/10.1002/jmv.25727