Serological tests based on viral glycoproteins for detecting neutralizing antibodies to measles, mumps and rubella viruses

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Introduction

Measles, mumps, and rubella viruses are highly contagious viruses that can cause serious clinical outcomes, complications, and death. Measles, mumps and rubella remain endemic in many parts of the world, and imported cases of measles lead to large outbreaks in communities with low vaccination coverage [1]. Before vaccines for these diseases were developed, there were approximately 30 million cases of measles and 2.6 million deaths from measles worldwide each year; the incidence of rubella was 100–1,000 cases per 100,000 people; and the prevalence of congenital rubella syndrome (CRS) was approximately 0.1–0.2 cases per 1,000 newborns, with the number of cases increasing to 0.8–4.0 per 1,000 newborns during epidemics, which historically occurred every 5–9 years [2].

Measles and mumps are RNA viruses of the family Paramyxoviridae, subfamily Paramyxoviridae; measles belongs to the genus Morbillivirus, and mumps belongs to the genus Rubulavirus. The rubella virus is also an RNA virus, but belongs to the family Matonaviridae, genus Rubivirus. These three viruses, despite differences in their families and genera, are often considered together because the epidemiology of the infections they cause in humans and the preventive measures used against them are similar [3].

Complications of measles include blindness, severe diarrhea, seizures, otitis, and pneumonia [4]. In rare cases, complications related to the central nervous system may develop. For example, acute disseminated encephalomyelitis occurs in approximately 1 case per 1,000 measles patients; and subacute sclerosing panencephalitis (SSPE) is extremely rare (about 1 case per 10,000–100,000 people who have had measles) [5].

Complications of mumps include meningoencephalitis, as well as orchitis and oophoritis in post-pubertal individuals. Mumps remains a common disease in many parts of the world, despite the availability of an effective vaccine.

Rare complications of rubella include thrombocytopenic purpura [6], arthritis-like diseases [7], encephalitis, and meningoencephalitis [8]. The rubella virus has a strong teratogenic effect: unvaccinated pregnant women who contract rubella in the first trimester have a 90% chance of giving birth to a child with CRS [9]. The most common manifestations of CRS are deafness, cataracts, blindness, congenital heart defects, endocrinopathies, microcephaly, encephalopathy, and developmental delay. Rubella infection in the first trimester can lead to stillbirth or miscarriage [10].

Historically, measles, rubella, and mumps were reliably diagnosed clinically and, in many cases, without the necessity for laboratory testing, as most doctors were familiar with the manifestations of these diseases [11].

The measles virus genome is a single-stranded, non-segmented RNA with a size of 15,894 nucleotides. The genome contains 6 genes that encode 8 viral proteins (6 structural and 2 non-structural). The structural protein hemagglutinin (HA) is the main antigenic determinant and has a highly conserved epitope. Hemagglutinin binds to cell receptors and is responsible for the adsorption of the virus to cells. Another envelope protein is the fusion protein (F), which mediates the fusion of the viral envelope with the plasma membrane of the host cell, thereby allowing the ribonucleoprotein complex (RNP) to enter the cytoplasm [12].

Mumps is a highly contagious paramyxovirus that is endemic in most regions of the world and continues to cause outbreaks even among populations with high immunization coverage. Mumps outbreaks in countries with high mumps vaccination coverage are explained by waning immunity and antigenic differences between vaccine strains and circulating wild-type viruses [13].

The mumps virus is an enveloped negative-strand RNA virus consisting of 15,384 nucleotides encoding seven genes. Two surface glycoproteins (GP) – the HA-neuraminidase protein (HN) and the F protein – are responsible for virus adsorption and virion membrane fusion with the host cell membrane, respectively, but both are required for cell-to-cell fusion. Virus-neutralizing antibodies are formed against these proteins, while virus-specific antibodies formed against the other 5 proteins located inside the virion do not have protective activity [14].

The vaccine strains currently in use originated in the mid-20th century and are phylogenetically distant from the viruses currently circulating [15].

Based on the diversity of small hydrophobic protein (SH) sequences, the mumps virus is classified into 12 genotypes, designated A–N. HN is the main target of humoral immunity during mumps virus infection. The gradual evolution of mumps viruses, rather than belonging to a specific genotype, leads to antigenic divergence from vaccine strains, which reduces the neutralizing ability of vaccine-induced antibodies [16].

The rubella virus genome consists of single-stranded non-segmented (+)RNA. The main GP proteins of the rubella virus envelope are the structural proteins E1 and E2. The E1 protein is responsible for the fusion of the viral envelope with the cell membrane, while the E2 protein is responsible for the attachment of the virus to cell receptors. The E1 protein is the main antigenic determinant and contains epitopes associated with hemagglutination and the formation of neutralizing antibodies [17–19].

A significant increase in the incidence of both measles and, to a lesser extent, rubella threatens the progress made towards the goal of eliminating these infections. Diagnosis of measles and rubella based solely on clinical signs is unreliable due to the nonspecific nature of the symptoms, the presence of mild forms of the disease, and the possibility of asymptomatic infection. The low prevalence of these infections has led to a decline in awareness of them, as well as an increase in the number of healthcare workers who lack experience in diagnosing rubella and measles. In such circumstances, laboratory tests become an indispensable tool for making a correct diagnosis and responding quickly to outbreaks of infection.

Despite the introduction of vaccines against measles, mumps, and rubella, cases of these diseases continue to be reported, indicating the necessity for reliable diagnostic tools for the timely detection and control of virus spread. Traditional diagnostic methods, such as serological tests, often face problems of specificity and sensitivity. This leads to possible false positive and false negative results and causes cross-immunoreactivity, which in turn complicates epidemiological monitoring and treatment.

The gold standard for detecting neutralizing antibodies to measles, mumps, and rubella viruses are neutralization assays (NA) on virus-sensitive cell cultures, plaque inhibition assays, and fluorescent antibody assays against viral membrane GP. However, these tests are technically difficult to perform, expensive, and take a long time. Commercial test systems based on enzyme-linked immunosorbent assay (ELISA) are most commonly used to detect IgG antibodies to measles, mumps, and rubella viruses. Unfortunately, they do not allow the detection of the main protective antibodies – neutralizing antibodies. Furthermore, many researchers have established the presence of cross-immunoreactivity in most commercial ELISA test systems [20].

The passive hemagglutination reaction (PHAR) is a very simple and inexpensive serological reaction used to detect neutralizing antibodies to measles, mumps, and rubella viruses. PHAR is based on the interaction of antibodies with their homologous viral antigens, resulting in the inability of red blood cells to settle. Thus, this reaction can be used to detect both antibodies and antigens. PHAR, in which viral GP, which induces neutralizing antibodies, is used as a viral antigen, is called gpPHAR and can be successfully used to determine immunogenicity to measles, mumps, and rubella when detecting neutralizing antibodies in vaccinated and recovered children and adults.

The aim of the study is to develop gpPHAR serological tests for the determination of neutralizing antibodies to measles, mumps, and rubella viruses.

Materials and methods

Cell cultures. Cell cultures from the I.I. Mechnikov Research Institute of Virology and Serology collection were used: HEL-3 – a strain of diploid human embryonic lung cells; Vero CCL-81 – a line of green monkey kidney cells; Vero E-6 – a cloned variant of the Vero cell line; Vero ECC – Vero cells from the European cell culture collection; A₅₄₉ – a line of transducible human lung adenocarcinoma cells; OCC/A-431 – a line of transducible human skin cancer cells; MeWo – a line of transducible human melanoma cells; BHK-F – a transducible cell culture of Syrian hamster kidney (France); PT – a line of transducible porcine testicular cells; MSC-16 – a variant of human mesenchymal stem cells. Cell cultures were grown on DMEM/F-12 medium with 10 mM HEPES, supplemented with 5 or 10% bovine serum (BIOCHEMSERVICE LLC, Vladimir, Russia) and 40 μg/ml gentamicin.

Viruses. Cold-adapted strains were used for replication in diploid HEL-3 cells at low temperature (30 °C): Leningrad-16-CA (L-16-CA) – domestic vaccine strain of measles virus; EP-6-CA – domestic vaccine strain of mumps virus; RA 27/3-CA – foreign vaccine strain of rubella virus; vRub-Ant-CA – domestic vaccine strain of rubella virus.

Cold-adapted vaccine strains of measles, mumps, and rubella viruses were created at the I.I. Mechnikov Research Institute of Vaccines and Sera (Method for obtaining a four-component live culture vaccine against measles, chickenpox, mumps, and rubella. Patent for invention No. 2693440, priority February 21, 2019, authors: V.V. Zverev, F.G. Nagieva, E.P. Barkova, O.V. Osokina).

Immune sera and monoclonal antibodies (mAbs). Human and animal immune sera against measles, mumps, rubella and other viruses were obtained from the collections of the viral infection diagnostics laboratory of the I.I. Mechnikov Federal State Budgetary Scientific Institution Research Institute of Viral Infections and the Moscow Scientific and Practical Center for Laboratory Research of the Moscow City Health Department. Monoclonal antibodies to L-16-CA strain of the measles virus and to the GP E1 strain Chendehill (C-74) RV of the rubella virus were obtained in the laboratory of the I.I. Mechnikov Research Institute of Virology and Immunology using hybridoma technology [21, 22].

Determination of the infectious activity of viruses. The infectious activity of measles, mumps, and rubella viruses was determined on Vero CCL-81 or Vero E-6 cell cultures grown on 24-well plates (Korea). The cell seeding concentration was 1 × 10⁵ cells per well. Virus-containing fluid (VCF) in a dilution of 10⁻¹ to 10⁻¹⁰ was added at 0.2 mL to the wells of the plates with the cell test culture for 1.0–1.5 hours at a temperature of 36.5 °C. Then, 0.8 mL of DMEM maintenance medium with increased glucose content and without serum was added to all wells. The plates were incubated at 35 °C and 5% CO₂. The results of mumps and rubella virus titration were recorded on the 7th day after infection in the hemadsorption reaction. The virus titer was taken as the maximum dilution of the virus causing hemadsorption in 50% of the wells with infected cell cultures in the absence of hemadsorption in the control wells. The measles virus titer was determined by cytopathic effect (CPE₅₀/0.2 mL) on infected cells, and the results were recorded on the 10th day.

Setting up the hemadsorption reaction. A 0.25% suspension of defibrinated guinea pig erythrocytes contained in Alsever's solution was prepared by washing the erythrocytes three times with 0.9% saline solution. 0.3 mL of the erythrocyte suspension was added to the wells of a plate containing infected and uninfected control cells, previously washed with phosphate-buffered saline (PBS). The cells with erythrocytes were kept for 30 minutes at 4 °C and for an additional 30 minutes at room temperature. Then, the wells of the plate were washed three times with 0.9% saline solution to remove erythrocytes, and the presence or absence of haemadsorption was recorded under a light microscope.

Virus neutralization test. VNT was performed on Vero CCL-81 cell culture using the previously described method [23].

Preparation of virus-specific glycoproteins. The following cell cultures were used to prepare virus-specific GP from VCF: for measles virus – Vero CCL-81, Vero ECC, HEL-3; for mumps viruses – Vero CCL-81, A₅₄₉, OCC/A, MSC-16, PT; for rubella virus – Vero E6, BHK-F (France), A₅₄₉, MSC-16.

Sensitive cell cultures were grown in flasks with an area of 175 cm². They were then infected with VCF and kept in a thermostat at 35 °C for 7–8 days until 70–90% of the cells were destroyed. The cells were then frozen at −70 °C for at least 24 hours, thawed, and centrifuged at 1500 rpm for 20 minutes. A sucrose-gelatin stabilizer was added to the supernatants, which were stored at −70 °C. To isolate viral GP, VCF, phytohemagglutinin (PHA) at a concentration of 25 μg/ml, and a 50% suspension of sheep erythrocytes, defibrinated and washed three times with 0.9% saline solution, were mixed in a 1 : 10 volume ratio, respectively. The mixture was incubated at 4 °C for 20 hours with periodic stirring to effectively bind GP to PHA on the surface of erythrocytes. The suspension was then centrifuged at 1500 rpm for 20 min. Saline solution was added to the resulting precipitate, consisting of formalinized sheep erythrocytes loaded with viral GP. Elution of viral GP from the surface of erythrocytes was performed at 37 °C for 1 hour. Erythrocytes were removed from the suspension by centrifugation at 1500 rpm for 20 minutes. To increase the stability of the obtained viral GP, bovine serum albumin (BSA) was added until a concentration of 1% was reached.

Determination of the diagnostic titer of viral glycoproteins in the hemagglutination assay (HA). 50 μL of PBS was added to each well of a 96-well V-shaped plate. Next, 50 μL of viral GP was added to the first well and titrated in two steps. Then, 50 μL of a 0.4% suspension of human erythrocytes (blood group 1 (0) Rhesus+) or guinea pig erythrocytes was added to all wells. After the erythrocytes settled in the control wells in the form of a “button” at the bottom of the well, the results were recorded. The titer of viral GP was determined by the last dilution where the erythrocytes were arranged in the form of an umbrella (across the entire bottom of the well) and expressed in hemagglutinating units (HAU).

Obtaining erythrocyte antigen diagnosticums based on sheep erythrocytes sensitized with viral glycoproteins. Sensitization of formalinized erythrocytes was performed using GP of measles, mumps, and rubella. For this purpose, the erythrocyte mass was resuspended in a 10-fold volume of cooled saline solution. It was precipitated by centrifugation at 1500 rpm for 20 min and a 50% concentration of erythrocyte suspension in saline solution was prepared. It was then mixed with a solution of viral GP, 0.33% chromium chloride (CrCl₃), and bidistilled water in a ratio of 1 : 1 : 1 : 10. The resulting mixture was incubated in a water bath at 42 °C for 1 hour. Next, an equal volume of saline solution was added and centrifuged at 1500 rpm for 5 minutes. The resulting precipitate, consisting of sensitized erythrocytes, was resuspended in saline solution enriched with 1% BSA and washed twice with the same solution. For gpPHAR, a 2.5% suspension of sensitized formalinized erythrocytes in saline solution with 1% BSA was prepared.

Immunoenzymatic analysis was performed using a standard method. In the first stage, 50 μL of antigen solution (GP (gpELISA) or VCF (ELISA)) was added to each well, incubated overnight at 4 °C, then the wells of the plate were blocked with a casein-sucrose solution for 90 min and the plates were dried for 2 h in a thermostat at 37 °C with the door open. To perform ELISA, 50 μL of dilutions of human, mouse, or guinea pig immune serum samples were added to the wells in PBS with Tween (PBS-T) in a two-step process, starting with a dilution of 1 : 100, and incubated for 90 min at 37 °C; The plate was washed 3 times with PBS-T; 50 μL of antivaccine conjugate solution (BioRad) in PBS-T + 1% BSA was added, and the plates were incubated for 60 min at 37 °C. the plate was washed three times with PBS-T; a solution of tetramethylbenzidine was added, incubated for 15 min in a dark place; the reaction was stopped with sulfuric acid, the results were recorded on a spectrophotometer at a wavelength of 450 nm (comparison wavelength 630 nm).

Serological method for gpPHAR testing to determine neutralizing antibodies in immune sera against measles, mumps, and rubella viruses. The gpPHAR test systems for detecting neutralizing antibodies to measles, mumps, and rubella viruses in human and animal sera are prepared on the basis of formalin-treated defibrinated sheep erythrocytes sensitized with measles, mumps, and rubella viruses. The method is described in detail for the detection of neutralizing antibodies in immune sera against varicella and herpes zoster viruses [23].

Statistical methods. GraphPad Prism 10 software packages were used for statistical processing of the results.

Results

To obtain GP of vaccine strains of measles, mumps, and rubella viruses, HEL-3, Vero CCL-81, Vero E-6, A₅₄₉, OCC/A-431, MeWo, BHK-F, MSC-16, and PT cell cultures were used. These cell cultures were infected with vaccine strains of measles, mumps, and rubella with a multiplicity of infection (MOI) of 0.01. The infectious titers of VCF after completion of virus replication in cell cultures were determined by titration using the limit dilution method on two cell cultures: HEL-3 (measles and mumps) and Vero CCL-81 (measles, mumps, and rubella viruses). Table 1 shows the results of titration for the hemadsorbing activity of viruses using a 0.25% suspension of human erythrocytes of blood group 1, Rhesus (+) and according to the CPE (measles virus).

 

Table 1. Titers in HAdU₅₀/0.2 mL and CPE_50/0.2 mL of virus-containing fluids obtained from cell cultures infected with vaccine strains of measles, mumps and rubella and titrated on Vero CCL-81 and HEL-3 cells

Таблица 1. Титры в ГАДЕ₅₀/0,2 мл и ЦПД₅₀/0,2 мл ВСЖ, полученных из клеточных культур, инфицированных вакцинными штаммами кори, ЭП и краснухи и титрованных на клетках Vero CCL-81 и ЛЭЧ-3

Virus-containing fluid from infected cells ВСЖ из инфицированных клеток	Virus titers in log10 HAdU50/0.2 mL and in CPE50/0.2 mL Титры вирусов в lg ГАДЕ50/0,2 мл и в ЦПД50/0,2 мл
	L-16-CА / Л-16-ХА		EP-6-CА / ЭП-6-ХА		RA 27/3-CА / RA 27/3-ХА
	Vero CCL-81*	ЛЭЧ-3 / HEL-3**	Vero CCL-81*	ЛЭЧ-3 / HEL-3*	Vero CCL-81*
Vero CCL-81	3.50 ± 0	4.50 ± 0	10.0 ± 0	6.50 ± 0	7.50 ± 0
A549	4.0 ± 0	4.0 ± 0	8.0 ± 0	7.50 ± 0	6.50 ± 0
OCC/A-431 / ОКК/А-431	4.75 ± 0	4.25 ± 0	8.0 ± 0	8.0 ± 0	6.0 ± 0
HEL-3 / ЛЭЧ-3	4.50 ± 0	4.50 ± 0	8.50 ± 0	8.0 ± 0	6.0 ± 0
MeWo	5.50 ± 0	5.00 ± 0	N/A / Н.и.	N/A / Н.и.	N/A / Н.и.
MSC-16 / МСК-16	N/A / Н.и.	N/A / Н.и.	8.0 ± 0	7.50 ± 0	7.50 ± 0
PT / ПТП	0	0	4.0 ± 0	4.0 ± 0	0

Note. * – titration by hemadsorption reaction (log10 HAdU₅₀/0.2 mL); ** – titration by cytopathic effect (CPE₅₀/0.2 mL); N/A – not assessed.

Примечание. * – титрование по реакции гемадсорбции (lg ГАДЕ₅₀/0,2 мл); ** – титрование по цитопатическому действию (ЦПД₅₀/0,2 мл); Н.и. – не исследовалось.

 

The results of titration of VCF on Vero CCL-81 and HEL-3 cell cultures, evaluated by the hemadsorption reaction, show that the L-16-CA strain of the measles virus, the EP-6-CA strain of the mumps virus, and the RA 27/3-CA strain of the rubella virus are effectively reproduced in all cell cultures used, with the exception of the PT cell culture. At the same time, measles and rubella viruses do not have membrane viral receptors to PT cells, and the mumps virus reproduces poorly in PT cells. It should also be noted that the measles virus is poorly detected when titrated on Vero CCL-81 cells by the hemadsorption reaction with human erythrocytes, since it is known that the hemagglutinin of the measles virus binds most effectively to the erythrocytes of rhesus macaque monkeys.

To create a gpPHAR serological test for the detection of neutralizing antibodies in human and animal immune sera to measles, mumps, and rubella viruses, it is necessary to determine their GP titers. The GP titers of the L-16-CA vaccine strain of the measles virus, the EP-6-CA vaccine strain of the mumps virus, and the RA 27/3-CA vaccine strain of the rubella virus were determined by HA with a 0.4% suspension of human erythrocytes. To confirm the reproducibility of the results, titration was performed in 3–4 replicates, and there were no deviations in the titration indicators. The results of the study are presented in Table 2.

 

Table 2. Titers of glycoproteins of vaccine strains of measles, mumps and rubella viruses established by hemagglutination assay (HA)

Таблица 2. Титры гликопротеинов вакцинных штаммов вирусов кори, эпидемического паротита и краснухи, установленных по РГА

Virus-containing fluid from cell cultures ВСЖ из культур клеток	Glycoprotein titers of measles, mumps and rubella vaccine strains isolated from infected cell cultures Титры гликопротеинов из вакцинных штаммов кори, паротита и краснухи, выделенных из инфицированных клеточных культур
	L-16-CА / Л-16-ХА	EP-6-CА / ЭП-6-ХА	RA 27/3-CА / RA 27/3-ХА
Vero CCL-81	1 : 8 ± 0	1 : 16 ± 0	1 : 16 ± 0
Vero E-6	N/A / Н.и.	N/A / Н.и.	1 : 16 ± 0
А549	1 : 16 ± 0	1 : 64 ± 0	1 : 32 ± 0
OCC/A-431 / OКК/А-431	1 : 8 ± 0	1 : 64 ± 0	1 : 32 ± 0
MSC-16 / МСК-16	1 : 4 ± 0	1 : 8 ± 0	1 : 8 ± 0
MeWo	1 : 16 ± 0	N/A / Н.и.	N/A / Н.и.
PT / ПТП	N/A / Н.и.	1 : 16 ± 0	< 2 ± 0

Note. N/A – not assessed.

Примечание. Н.и. – не исследовалось.

 

Further in the study, we used measles virus GP obtained from infected MeWo cell culture, for mumps – GP from infected A₅₄₉ cell culture, and for rubella diagnosticum – GP from infected Vero E-6 cell culture.

Table 3 shows the results of comparative titration of human immune serum and guinea pig immune serum in VNT and gpPHAR.

 

Table 3. Comparative titration of human and guinea pig immune sera to measles, mumps and rubella viruses in VNT (titers in HAdU₅₀/0.2 mL and CPE₅₀/0.2 mL) and in gpPHAR (titer in HAU₅₀/0.5 mL)

Таблица 3. Сравнительное титрование иммунных сывороток человека и морской свинки к вирусам кори, ЭП и краснухи в РН (титры в ГАДЕ₅₀/0,2 мл и ЦПД₅₀/0,2 мл) и в gpРПГА (титр в ГАЕ₅₀/0,5 мл)

Immune sera Иммунные cыворотки	Comparative titers of immune sera in two serological reactions Сравнительные титры иммунных сывороток в двух серологических реакциях
	VNT in HAdU50/0.2 mL РН в ГАДЕ50/0,2 мл			gpPHAR in HA50/0.5 mL gpРПГА в ГАЕ50/0,5 мл
	L-16-CА* Л-16-ХА	EP-6-CА ЭП-6-ХА	RA 27/3-CА RA 27/3-ХА	L-16-CА Л-16-ХА	EP-6-CА ЭП-6-ХА	RA 27/3-CА RA 27/3-ХА
Human immune serum Иммунная сыворотка человека	1 : 800 ± 0	1 : 3200 ± 0	1 : 800 ± 0	1 : 800 ± 0	1 : 3200 ± 0	1 : 800 ± 0
Guinea pig immune serum Иммунная сыворотка морской свинки	1 : 1600 ± 0	1 : 3200 ± 0	1 : 1600 ± 0	1 : 1600 ± 0	1 : 3200 ± 0	1 : 1600 ± 0
Non-immune human serum immune human serum Неиммунная сыворотка человека	0	0	0	< 2	< 2	< 2
Non-immune guinea pig serum Неиммунная сыворотка морской свинки	0	0	0	< 2	< 2	< 2

Note. * – titer assessment in CPE₅₀/0.2 mL.

Примечание. * – оценка титра в ЦПД₅₀/0,2 мл.

 

These results showed that in VNT with a 1000 dose of measles, mumps, and rubella viruses, and in gpPHAR using antigenic diagnostic reagents on sensitized GP formalinized sheep erythrocytes, the titers do not differ, and the level of neutralizing antibodies detected in immune sera is directly dependent on the titer of viral GP.

According to foreign authors, the main disadvantage of commercial test systems, in particular in ELISA serological reactions, is the presence of cross-immunoreactivity.

In order to determine the presence or absence of cross-immunoreactivity when using the gpPHAR serological test, experiments were conducted with homologous and heterologous virus sera.

Fig. 1 shows the gpPHAR titers of immune sera from humans, guinea pigs, horses, and mice mAb-221 and mAb-96 homologous to the vaccine strain L-16-CA to the hemagglutinin of the L-16 strain of the measles virus. Immune sera from humans and guinea pigs to vaccine strains EP-6-CA and RA 27/3-CA, respectively, mumps and rubella viruses, as well as guinea pig immune sera to the A169 strain of cytomegalovirus, guinea pig immune serum to the vZelVax vaccine strain of herpes zoster virus.

 

Fig. 1. Titers in gpPHAR of homologous and heterologous immune sera and monoclonal antibodies to the measles vaccine strain L-16-CA. 1 – human serum to measles virus; 2 – guinea pig serum to measles virus; 3 – horse serum to measles virus; 4 – mouse mAb-221 to hemagglutinin of measles virus; 5 – mouse mAb-96 to hemagglutinin of measles virus; 6 – human serum to mumps virus; 7 – human serum to rubella virus; 8 – guinea pig serum to cytomegalovirus; 9 – human serum to herpes zoster virus; 10 – diagnostic control.

Рис. 1. Титры в gpРПГА гомологичных и гетерологичных иммунных сывороток и моноклональных антител к вакцинному штамму Л-16-ХА вируса кори. 1 – сыворотка человека к вирусу кори; 2 – сыворотка морской свинки к вирусу кори; 3 – сыворотка лошади к вирусу кори; 4 – МКА-221 к гемагглютинину вируса кори; 5 – МКА-96 к гемагглютинину вируса кори; 6 – сыворотка человека к вирусу эпидемического паротита; 7 – сыворотка человека к вирусу краснухи; 8 – сыворотка морской свинки к цитомегаловирусу; 9 – сыворотка человека к вирусу опоясывающего герпеса; 10 – контроль диагностикума.

 

The results clearly demonstrate that the constructed gpPHAR does not possess spontaneous hemagglutination, detects neutralizing protective antibodies in humans and animals against the L-16-CA vaccine strain of the measles virus, and does not possess cross-immunoreactivity with immune sera against other viral agents.

Fig. 2 shows the results of titration in gpPHAR of immune sera from humans, guinea pigs, and goats homologous to the EP-6-CA vaccine strain. Immune sera from humans to the vaccine strain vZelVax of the herpes zoster virus, guinea pig serum to the vaccine strain vZelVax of the herpes zoster virus, guinea pig serum to the RA 27/3-CA vaccine strain of rubella virus, serum to the domestic Rub-Ant vaccine strain of rubella virus, guinea pig serum to the L-16-CA vaccine strain of measles virus.

 

Fig. 2. Titers in gpPHAR of homologous and heterologous immune sera to the vaccine strain EP-6-CA of the mumps virus. 1 – human serum to mumps virus; 2 – guinea pig serum to mumps virus; 3 – goat serum to mumps virus; 4 – human serum to human herpes zoster virus; 5 – guinea pig serum to herpes zoster virus; 6 – guinea pig serum to the rubella virus strain RA 27/3-CА; 7 – guinea pig serum to the rubella virus strain Rub-Ant; 8 – guinea pig serum to measles virus; 9 – diagnostic control.

Рис. 2. Титры в gpРПГА гомологичных и гетерологичных сывороток к вакцинному штамму ЭП-6-ХА вируса эпидемического паротита. 1 – сыворотка человека к вирусу эпидемического паротита; 2 – сыворотка морской свинки к вирусу эпидемического паротита; 3 – сыворотка козы к вирусу эпидемического паротита; 4 – сыворотка человека к вирусу опоясывающего герпеса человека; 5 – сыворотка морской свинки к вирусу опоясывающего герпеса; 6 – сыворотка морской свинки к штамму RA 27/3-ХА вируса краснухи; 7 – сыворотка морской свинки к штамму Rub-Ant вируса краснухи; 8 – сыворотка морской свинки к вирусу кори; 9 – контроль диагностикума.

 

The figure clearly demonstrates the absence of cross-immunoreactivity of immune sera in the gpPHAR serological test with an antigenic diagnosticum based on the mumps virus GP.

Fig. 3 shows the results of titration in gpPHAR of homologous immune sera from humans, goats, and guinea pigs to the RA 27/3-CA vaccine strain of the rubella virus and mouse mAbs to the E1 glycoprotein of the Chendehill (C-74) RV strain of the rubella virus. Heterologous immune sera include human and guinea pig sera to vaccine strains of measles, mumps, and herpes zoster viruses, and guinea pig serum to cytomegalovirus.

 

Fig. 3. Titers in gpPHAR of homologous and heterologous immune sera to the rubella virus strain RA 27/3-CA. 1 – human serum to rubella virus; 2 – goat serum to rubella virus; 3 – murine mAb-257 to rubella virus glycoprotein E1; 4 – human serum to the strain L-16-CA of measles virus; 5 – guinea pig serum to the strain L-16-CA of measles virus; 6 – human serum to the EP-6-CA strain of mumps virus; 7 – guinea pig serum to the EP-6-CA strain of mumps virus; 8 – guinea pig serum to cytomegalovirus; 9 – guinea pig serum to herpes zoster virus; 10 – diagnostic control.

Рис. 3. Титры в gpРПГА гомологичных и гетерологичных иммунных сывороток к штамму RA 27/3-ХА вируса краснухи. 1 – сыворотка человека к вирусу краснухи; 2 – сыворотка козы к вирусу краснухи; 3 – МКА-257 к гликопротеину Е1 вируса краснухи; 4 – сыворотка человека к штамму Л-16-ХА вируса кори; 5 – сыворотка морской свинки к штамму Л-16-ХА вируса кори; 6 – сыворотка человека к штамму ЭП-6-ХА вируса паротита; 7 – сыворотка морской свинки к штамму ЭП-6-ХА вируса паротита; 8 – сыворотка морской свинки к цитомегаловирусу; 9 – сыворотка морской свинки к вирусу опоясывающего герпеса; 10 – контроль диагностикума.

 

The results of titration of immune sera in gpPHAR allow us to conclude that the constructed antigenic diagnosticum based on GP E1 of the RA 27/ vaccine strain 3-CA strain of the rubella virus does not cause spontaneous hemagglutination and does not possess cross-immunoreactivity, which is particularly important for making a correct diagnosis and assessing the quality of vaccine preparations based on the titers of induced neutralizing antibodies.

It is known that Vero CCL-81 and Vero ECC cell cultures are highly sensitive to the measles virus. GP was extracted from cell cultures infected with the L-16-CA vaccine strain of the measles virus using PHA lectin. Based on the isolated GP, antigenic diagnostic reagents were prepared on formalinized sheep erythrocytes for gpPHAR titration of 14 immune sera from children who had had measles infection. It was shown that the titers of neutralizing antibodies in children who had had measles infection, detected in gpPHAR by two antigenic diagnostics, differed by only one two-fold step.

Furthermore, blood serum samples from 11 patients with suspected mumps were examined in three serological tests in gpPHAR, gpELISA, and ELISA.

Analysis of the results presented in Table 4 shows that all 11 immune sera tested were positive for mumps in all three serological tests. The results demonstrate the high sensitivity of gpPHAR: in most cases, the titers of samples in gpPHAR were higher than in gpELISA and ELISA.

 

Table 4. Comparative serum titers of patients with suspected mumps infection, determined in 3 serological tests

Таблица 4. Сравнительные титры сывороток пациентов с подозрением на паротитную инфекцию, поставленную в 3 серологических тестах

Immune sera Иммунные cыворотки	Serum titers in various serological tests Титры сывороток в различных серологических тестах
	gpPHAR gpРПГА	gpELISA gpИФА	ELISA ИФА
№ 1	1 : 3200	1 : 200	1 : 800
№ 2	1 : 3200	1 : 3200	1 : 6400
№ 3	1 : 6400	1 : 200	1 : 400
№ 4	1 : 800	1 : 3200	1 : 400
№ 5	1 : 3200	1 : 3200	1 : 800
№ 6	1 : 6400	1 : 400	1 : 200
№ 7	1 : 3200	1 : 3200	1 : 800
№ 8	1 : 3200	1 : 200	1 : 800
№ 9	1 : 3200	1 : 3200	1 : 400
№ 10	1 : 1600	1 : 1600	1 : 1600
№ 11	1 : 6400	1 : 1600	1 : 400

 

Fig. 4 shows the results of titration in HAU₅₀/0.5 mL in gpPHAR and gpELISA of immune sera from children who had had rubella before 2000, whose sera were stored at −20 °C.

 

Fig. 4. Determination of titers of immune sera in gpPHAR and gpELISA based on glycoproteins of the RA 27/3-CA strain rubella virus.

Рис. 4. Определение титров иммунных сывороток в gpРПГА и gpИФА на основе гликопротеинов штамма RA 27/3-ХА вируса краснухи.

 

Analysis of the titration results shows that predominantly immune sera have identical titers, sometimes differing by one titer step. In most cases, the titers in gpELISA were lower than those in gpPHAR, which confirms the high sensitivity of the developed test system.

Discussion

Serological surveillance of measles, mumps, and rubella infections, i.e., measuring the seroprevalence of antibodies to these infections, is an important tool for monitoring herd immunity, vaccination coverage and long-term vaccine-induced immunity, which allows for the identification of gaps in immunity and thus provides more rapid management of measles, mumps, and rubella outbreaks.

High-throughput, efficient, and accurate methods are necessary to assess complex immunity and facilitate rapid action to contain infectious outbreaks.

Neutralizing antibodies against measles, mumps, and rubella infections are a good indicator of clinical protection against these infections, but they are difficult to measure [24].

Therefore, the aim of this study was to develop a highly sensitive, specific, and simple serological test that does not cross-react with immune sera to other viral agents.

We have previously shown that the gpPHAR serological test fully meets these requirements for the detection of neutralizing antibodies in immune sera against varicella-zoster virus and herpes zoster [23].

In this study, we described the creation of three gpPHAR serological test systems used to evaluate neutralizing antibodies in immune sera against measles, mumps, and rubella viruses.

Highly sensitive cell cultures to the three viruses were identified, allowing viral GP to be extracted in titers sufficient for the creation of antigenic diagnosticums and their sensitization on formalinized sheep erythrocytes. Antigenic diagnosticums remain effective when stored at −70 °C until use.

A comparative titration of human and guinea pig immune sera to vaccine strains of L-16-CA measles virus, EP-6-CA mumps virus, and RA 27/3-CA rubella virus was performed in two serological reactions: VNT and gpPHAR. A 100% correlation of neutralizing antibody titers in immune sera against measles, mumps, and rubella was established (Table 3).

In the constructed gpPHAR serological tests, created on GP of measles, mumps, and rubella viruses sensitized on formalinized sheep erythrocytes to detect neutralizing antibodies, the absence of cross-immunospecficity was clearly established (Figs. 1–3).

Using three serological test systems (gpPHAR, gpELISA, and ELISA), comparative titration of immune sera from patients who had recovered from mumps was performed (Table 4). Homologous results positive for mumps were found in all three serological tests: gpPHAR, gpELISA, and ELISA.

Conclusion

An important component of the immune response is humoral immunity, which is provided by neutralizing antibodies. The advantage of gpPHAR is that it detects only protective neutralizing antibodies induced by viruses in both humans and animals. gpPHAR does not have cross-immunoreactivity, unlike immunoenzyme test systems; gpPHAR has high sensitivity and specificity; gpPHAR is a 100% reproducible test system and can be easily reproduced in any clinical laboratory.

About the authors

Firaya G. Nagieva

I.I. Mechnikov Research Institute of Vaccines and Sera

Author for correspondence.
Email: fgn42@yandex.ru
ORCID iD: 0000-0001-8204-4899

D. Sci. (Med.), Associate Professor, Head of Laboratory of Hybrid Cell Cultures, Department of Virology

Russian Federation, Moscow

Elena P. Barkova

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: e.barkova2012@yandex.ru
ORCID iD: 0000-0002-3369-8869

Cand. Sci. (Biol.), Leading Researcher, Laboratory of Hybrid Cell Cultures, Department of Virology

Russian Federation, Moscow

Olga S. Kharchenko

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: bio139@yandex.ru
ORCID iD: 0000-0002-2169-9610

Researcher, Laboratory of Genetics of DNA-Containing Viruses, Department of Virology

Russian Federation, Moscow

Alexander V. Sidorov

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: sashasidorov@yandex.ru
ORCID iD: 0000-0002-3561-8295

Cand. Sci. (Biol.), Head of Laboratory of Genetics of DNA-Containing Viruses, Department of Virology

Russian Federation, Moscow

Natalia N. Vlasova

Federal Scientific Center – All-Russian Research Institute of Experimental Veterinary Medicine named after K.I. Skryabin and Ya.O. Kovalenko, Russian Academy of Sciences

Email: vlanany@yandex.ru
ORCID iD: 0000-0001-8707-7710

D. Sci. (Biol.), Principal Senior Research Scientist of Laboratory of Biochemistry and Molecular Biology

Russian Federation, Moscow

Olga A. Trubacheva

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: trolana@mail.ru
ORCID iD: 0009-0005-0821-5553

Leading Specialist, Laboratory of Hybrid Cell Cultures, Department of Virology

Russian Federation, Moscow

Yulia N. Tarakanova

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: ytarakanova@mail.ru
ORCID iD: 0000-0003-3226-5989

Cand. Sci. (Biol.), Head of Laboratory of Diagnostics of Viral Infections, Department of Virology

Russian Federation, Moscow

Vladislav V. Semerikov

Federal State Budgetary Educational Institution of Higher Education Perm State Pharmaceutical Academy of the Ministry of Health

Email: metodkkikb1@yandex.ru
ORCID iD: 0000-0002-5346-8104

MD, Professor of the Department of Extreme Medicine and Commodity Science, Chief Freelance Epidemiologist at the Ministry

Russian Federation, Perm

Alfiya K. Tagirova

First Moscow State Medical University named after I.M. Sechenov of the Ministry of Health of the Russian Federation (Sechenov University)

Email: tagirova_ak@staff.sechenov.ru
ORCID iD: 0000-0001-9286-687X

PhD, Associate Professor at the Department of Biological Chemistry

Russian Federation, Moscow

Dinora A. Yakovleva

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: dyakovleva1610@yandex.ru
ORCID iD: 0000-0001-8771-4177

Senior Researcher, Laboratory of Diagnostics of Viral Infections, Department of Virology

Russian Federation, Moscow

Evgeny A. Pashkov

I.I. Mechnikov Research Institute of Vaccines and Sera; First Moscow State Medical University named after I.M. Sechenov of the Ministry of Health of the Russian Federation (Sechenov University)

Email: pashckov.j@yandex.ru
ORCID iD: 0000-0002-5682-4581

PhD, Junior Researcher, Laboratory of Applied Virology; Senior Lecturer of Microbiology, Virology and Immunology Department

Russian Federation, Moscow; Moscow

Ekaterina S. Osipova

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: Osipova.e.s201@gmail.com
ORCID iD: 0009-0000-6365-7147

Laboratory Assistant Researcher of the Laboratory of Hybrid Cell Cultures, Department of Virology

Russian Federation, Moscow

Anastasiya M. Polisadova

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: polisadova.an@gmail.com
ORCID iD: 0009-0003-5845-5059

Laboratory Assistant Researcher, Laboratory of Hybrid Cell Cultures, Department of Virology

Russian Federation, Moscow

Oksana A. Svitich

I.I. Mechnikov Research Institute of Vaccines and Sera; First Moscow State Medical University named after I.M. Sechenov of the Ministry of Health of the Russian Federation (Sechenov University)

Email: sviticgoa@yandex.ru
ORCID iD: 0000-0003-1757-8389

D. Sci. (Med.), Corresponding Member of the Russian Academy of Sciences, Director

Russian Federation, Moscow; Moscow

Vitaly V. Zverev

I.I. Mechnikov Research Institute of Vaccines and Sera; First Moscow State Medical University named after I.M. Sechenov of the Ministry of Health of the Russian Federation (Sechenov University)

Email: vitalyzverev@outlook.com
ORCID iD: 0000-0001-5808-2246

D. Sci. (Biol.), Professor, RAS Full Member, Head of Laboratory of Molecular Biotechnology

Russian Federation, Moscow; Moscow

References

Supplementary files