Riding the Omicron BA.5 Wave: Improved Humoral Response after Vaccination with Bivalent Omicron BA.4-5-Adapted mRNA SARS-CoV-2 Vaccine in Chronic Hemodialysis Patients

Hemodialysis patients faced an excess morbidity and mortality during the COVID-19 pandemic. We evaluated the effect of second-generation mRNA vaccines against Omicron BA.4 and BA.5 variants of SARS-CoV-2 on humoral immunity. The study population comprised 66 adult hemodialysis patients who have encountered four SARS-CoV-2 antigen contacts through vaccination or infection. We assessed their humoral response using an anti-SARS-CoV-2 spike receptor binding domain IgG antibody assay (S-RBD-ab), measuring neutralizing antibodies against ancestral strain of SARS-CoV-2, Delta, and Omicron in a surrogate virus neutralization test (SVNT), and specifically against BA.5 in a plaque reduction neutralization test (PRNT) before and four weeks after vaccination with Comirnaty Original/Omicron BA.4-5. During the following six months, SARS-CoV-2 infections and symptom severity were documented. The bivalent mRNA vaccine led to a 7.6-fold increase in S-RBD-ab levels and an augmented inhibition of the Omicron variant in SVNT by 35% (median). Seroconversion in the Omicron BA.5-specific PRNT was attained by in 78.4% of previously negative patients (29/37). Levels of S-RBD-ab correlated with inhibition in the Omicron-specific SVNT and neutralization titers in the BA.5-PRNT. Eleven SARS-CoV-2 infections occurred in the six-month follow-up, none of which took a life-threatening course. The bivalent mRNA vaccine improved the SARS-CoV-2 virus variant-specific humoral immunity in chronic hemodialysis patients. Measurement of S-RBD-ab can be used in hemodialysis patients to estimate their humoral immunity status against Omicron BA.5.


Introduction
Patients on maintenance dialysis show humoral and cellular immune deficiency [1][2][3], which renders them more prone to infections.It has been previously reported that dialysis patients have a reduced immune response to influenza or hepatitis B virus vaccines [4,5].
In the pre-vaccination era of the SARS-CoV-2 pandemic, it has been shown that COVID-19 disease claimed more victims among patients on dialysis compared to the general population [6].Continuous evolution of SARS-CoV-2 virus and progressive immunization of the population as a result of vaccination and/or infections changed the situation: the COVID-19 mortality of dialysis patients decreased from 20-30% to 2.2% [7].However, new challenges include a higher transmission rate of emerging virus variants and a reduced protection after vaccination with the wild-type mRNA vaccine [8,9].The second-generation mRNA vaccines were designed to be bivalent: 50% of the anti-wild-type strain and the adapted vaccine (firstly to the Omicron BA.1 variant and later to BA.4 and BA.5), each.In immunocompetent patients, this led to an improved humoral vaccination response [10,11].Its effect in hemodialysis patients was not evaluated in the approval studies and first publications.
In autumn 2022, strict containment strategies were reduced in Germany.Subsequently, a steep rise in incidence of SARS-CoV-2 infections was observed [12].The majority of our hemodialysis patients had received their last wild-type SARS-CoV-2 vaccination or became infected with this virus between February and April 2022.The Robert Koch Institute (the German government research institute responsible for infection disease control) recommended a booster with the bivalent mRNA vaccine for immunodeficient patients who already had four antigen contacts to the spike protein of SARS-CoV-2 (by either infection or vaccination), at least six months after the last one.This study was designed to explore the effect of a bivalent mRNA vaccine on the humoral immune response and clinical outcomes in chronic maintenance hemodialysis patients.

Study Design and Population
We investigated adult hemodialysis patients in tertiary care dialysis units at the outpatient KfH Dialysis Centers in Offenbach, Gross-Gerau, and at the Department of Nephrology and Dialysis of Sana Hospital Offenbach, Germany, in a prospective, investigator-initiated, non-interventional, observational trial.
Out of 207 patients treated in the three mentioned dialysis centers, we report on a subgroup of 66 patients who have had four exposures to the spike protein of SARS-CoV-2 by either infection or vaccination (Figure 1, patient recruitment).All enrolled patients were vaccinated with the adapted COVID-19 vaccine Comirnaty Original/Omicron BA.4-5 in October 2022, hereafter also referred to as fifth antigen contact.We assessed the humoral response to the vaccination before (t 0 ) and four weeks after the vaccination (t 1 ) with the bivalent mRNA vaccine (Figure 2).

Assessment of Anti-SARS-CoV-2 Spike-Specific Antibodies
Antibodies targeted against the SARS-CoV-2 spike receptor binding domain (S-RBDab) were measured using the Abbott SARS-CoV-2 IgG II Quant and SARS-CoV-2 IgG (Abbott GmbH, Wiesbaden, Germany) on the Abbott Alinity i platform.The cut-off for positivity was 7.1 BAU/mL (manufacturer's specification).To assess immunological protection against severe SARS-CoV-2 infection, we used the threshold for protection as 500 BAU/mL of the Delta variant [14].During the six months following this vaccination, SARS-CoV-2 infections and symptom severity according to the World Health Organization guidelines were documented [13].
In all centers, filtering face piece (FFP2) masks were still compulsory for patients and staff.

Assessment of Anti-SARS-CoV-2 Spike-Specific Antibodies
Antibodies targeted against the SARS-CoV-2 spike receptor binding domain (S-RBDab) were measured using the Abbott SARS-CoV-2 IgG II Quant and SARS-CoV-2 IgG (Abbott GmbH, Wiesbaden, Germany) on the Abbott Alinity i platform.The cut-off for positivity was 7.1 BAU/mL (manufacturer's specification).To assess immunological protection against severe SARS-CoV-2 infection, we used the threshold for protection as 500 BAU/mL of the Delta variant [14] CaCo-2 (human colon carcinoma) cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS), 100 IU/mL of penicillin, and 100 g/mL of streptomycin.All culture reagents were purchased from Sigma (St. Louis, MO, USA).The CaCo-2 cells were originally obtained from DSMZ (Braunschweig, Germany, no.: ACC 169), differentiated by serial passaging and selected for high permissiveness to virus infection [15].
Cultured Cancer Coli (CaCo)-2 cells were subsequently inoculated for 72 h with the prepared patients' sera/virus mix.Thereafter, CaCo-2 cells were analyzed for cytopathic effect (CPE) formation using light microscopy to define the neutralization titers as reciprocal value of the highest dilution of serum that prevented infection of 50% of the cells.The criteria for CPE are described in detail before [16].Each serum sample was tested in duplicate, in the case of discrepancies, and the lowest observed titer was chosen.Cut-off for positivity was set to ≥1:20 dilution.

Detection of SARS-CoV-2 Infections
Polymerase chain reaction (PCR) testing of nose and throat swabs was performed in case of symptoms suggesting respiratory infection.Only a fraction of the patients (Sana Hospital Offenbach, n = 11) was tested weekly using SARS-CoV-2 PCR.
The SARS-CoV-2 PCR test was performed as dual target PCR for ORF1 and E genes on a Cobas ® 6800/8800 system (ROCHE Diagnostics, Mannheim, Germany).

Study End Points
The primary end point of this study was the percentage of patients with a positive seroconversion in PRNT specific for Omicron BA.5 four weeks after the vaccination with the bivalent mRNA vaccine in the patient subgroup with negative PRNT prior to this booster.
Secondary end points were (a) the SARS-CoV-2 infection rate after the vaccination and (b) the occurrence of life-threatening COVID-19 disease and COVID-19-associated mortality despite five past exposures to SARS-CoV-2 during the six-month follow-up.

Statistics
Continuous variables were presented as medians and interquartile ranges (IQR) and categorical variables were reported as frequencies and percentages.To differentiate between patient cohorts, the Fisher's exact test for categorical variables was used.For quantitative variables, a t-test or a Mann-Whitney-U test was applied for parametric and nonparametric data, respectively.For comparison of more than two groups, one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc correction or the Kruskal-Wallis test followed by Dunn's post hoc test was performed for parametric and nonparametric data, respectively.For related samples comprising more than two groups, Friedman test was performed.With means of the Spearman's rank correlation analysis, bivariate relationships by calculating Spearman's Rho (correlation coefficient) were determined.Normality was assessed using the Kolmogorov-Smirnov test.To evaluate the determinants of independent predictors of response rate to vaccination, univariate logistic regression analyses were performed and odds ratios (OR) and corresponding 95% confidence intervals were calculated.
All p-values reported were two-sided, and the level of significance was set to p < 0.05.Statistical analyses were performed using GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA) and BiAS (v11.01;epsilon-Verlag, Nordhastedt, Germany).

Basic Study Cohort Characteristics
The demographic and clinical characteristics of the 66 enrolled patients are described in Table 1.The applied vaccines and infection status are shown in Table 2. Prior to the bivalent vaccine, 13 patients had experienced 14 SARS-CoV-2 infections.The SARS-CoV-2 Omicron BA.5 variant was dominant in Germany during the first half of this trial, followed by the XBB.1 sublines [17].BA.5-specific PRNT positivity was detected in 43.9% (29/66) of our patients before the vaccination, defined as a titer ≥1:20.Nineteen of these 29 patients (65.5%) had not previously been infected with SARS-CoV-2.Thus, they were able to neutralize Omicron BA.5 in PRNT after vaccination with monovalent BNT162b2 (Comirnaty) and/or mRNA-1273 (Moderna) vaccines developed against the wild-type virus, having had the last booster eight months ago.
However, the univariate logistic regression analysis revealed that patients who experienced a SARS-CoV-2 infection among their four previous antigen contacts had a twelve times higher chance for a positive result in the Omicron-SVNT and a four times higher chance for positivity in Omicron BA.5-specific PRNT compared to those patients without such an infection (odds ratios, OR 12.4, p = 0.003; OR 4.35, p = 0.028, respectively).

Humoral Response Four Weeks after the Vaccination (Time Point t 1 )
The primary end point of this trial, a positive seroconversion in the PRNT specific for Omicron BA.5, was attained by 78.4% (29/37) of the patient subgroup that was negative in the Omicron BA.5-PRNT before the vaccination.

Clinical Outcomes
The bivalent vaccine did not cause any moderate or severe adverse events (Grade 2-4 on the FDA toxicity grading scale).Mild reactions to the vaccine were not recorded.

Clinical Outcomes
The bivalent vaccine did not cause any moderate or severe adverse events (Grade 2-4 on the FDA toxicity grading scale).Mild reactions to the vaccine were not recorded.
In the first four weeks after the vaccination with bivalent mRNA vaccine, no symptomatic infections with SARS-CoV-2 occurred among the study participants despite high infection rates in the general population and reduction of the contact restrictions in public life (Figure A1).However, subsequently eleven SARS-CoV-2 infections occurred between four weeks and six months following the vaccination (secondary end point a).Ten patients showed mild symptoms and one patient suffered from moderate COVID-19 disease (pneumonia).This patient had a low neutralization titer in the PRNT (1:20).
Life-threatening COVID-19 disease, despite five past exposures to SARS-CoV-2 (secondary end point b), did not occur during the six-month follow-up.

Discussion
Studies on the first and second booster vaccinations against SARS-CoV-2 demonstrated how the S-RBD-ab level decreased rapidly after two to six months after vaccination [18][19][20][21].
Congruently, our findings show low S-RBD-ab level at the time point t 0 before the vaccination with the bivalent vaccine (six to eight months after the fourth SARS-CoV-2 antigen exposure).Nevertheless, the activity of the neutralizing antibodies was sufficient to prevent cytopathic effect in the Omicron BA.5-PRNT in 43.9% (29/66) of patients.One-third (34.5%) of those has had a previous breakthrough infection.Our data suggest that protection is more robust with the combination of repeated vaccinations and SARS-CoV-2 infection than with the same number of vaccinations alone.Consistent with our findings, Huth et al. [22] also reported significantly higher anti-spike IgG concentrations and neutralization activity in hemodialysis patients with breakthrough infections as compared to vaccination only.
The bivalent mRNA-vaccine against COVID-19 (Comirnaty Original/Omicron BA.4-5) led to an increase in S-RBD-ab levels and neutralizing capacity against Omicron, the prevalent SARS-CoV-2 subtype at the time of the study.The humoral immunity (assessed with neutralization tests) against wild-type SARS-CoV-2 and Delta variants was already present before the vaccination.The intention for the choice of the bivalent Omicron BA.4/BA.5-adaptedvaccines in the general population was to enhance neutralization breadth and to confer protection to individuals with no preexisting immunity against SARS-CoV-2 [23].In view of our data, probably the anti-wild-type strain vaccine component would not have been mandatory for our hemodialysis patients exposed to SARS-CoV-2 antigen (mostly to the wild-type) four times within 18 months.
A good correlation of the S-RBD-ab levels with a wild-type SARS-CoV-2 PRNT has already been reported [24,25].Espi et al. [26] established that S-RBD-ab ≥ 997 BAU/mL were systematically associated with positivity in PRNT against WT of SARS-CoV-2 in patients receiving maintenance hemodialysis.
Huth et al. [22] showed that the S-RBD-ab concentration after booster vaccination was the most important predictor of high neutralization activity against BA.4 and BA.5.Our data confirm that levels of S-RBD-ab correlate significantly with inhibition in the Omicronspecific SVNT and the titer of the Omicron BA.5-PRNT both before and four weeks after the vaccination.In our cohort, S-RBD-ab titer ≥2240.4BAU/mL after the vaccination with the bivalent Omicron BA.4-5-adapted mRNA vaccine were associated with positivity in the Omicron-SVNT and Omicron-BA.5-PRNT.
The limitation of our study is the rather small size of the cohort (66 participants, eleven SARS-CoV-2 infections in the study period).The homogeneity and stringent characterization of the studied patients (exactly five antigen contacts to the SARS-CoV-2 spike protein) represents a strength of the study.
Due to the progressive immunization of the population against SARS-CoV-2 through vaccination and infections, the COVID-19 pandemic is moving on to an endemic phase.Mortality from the disease induced by the currently circulating strains has substantially decreased; however, contagiousness has risen continuously.Thus, the immune-deficient dialysis patients' population remains endangered.It is also important to reduce SARS-CoV-2 transmission within the dialysis facilities to avoid supply shortages caused by simultaneous disease outbreaks among patients and staff.As a result of the high vaccination rate, there were no COVID-19 outbreaks in our dialysis centers during the autumn wave of 2022.

Conclusions
The bivalent vaccination improved the humoral IgG-related BA.4-5-specific immune response in more than half of the investigated hemodialysis patients.This translated in a low COVID-19 disease rate of mostly mild courses from four weeks after the bivalent vaccine onwards.
As there was a good correlation between variant-specific plaque reduction neutralization test results, ELISA-based variant-specific surrogate virus neutralization test, and SARS-CoV-2 wild-type antibody response as measured in immunoassay, one of the last two mentioned may be sufficient for clinical practice to estimate the patient's humoral immunity status even against the BA.5 variant.
Annually adapted SARS-CoV-2 vaccinations could be an instrument together with seasonal influenza vaccination to protect the vulnerable group of hemodialysis patients.

Figure 2 .
Figure 2. Flowchart of the study setup.CIHD = chronic intermittent hemodialysis, t0 = before the vaccination with Comirnaty Original/BA.4-5,six to eight months after the fourth antigen exposure to SARS-CoV-2, and t1 = four weeks after the vaccination.

Figure 2 .
Figure 2. Flowchart of the study setup.CIHD = chronic intermittent hemodialysis, t 0 = before the vaccination with Comirnaty Original/BA.4-5,six to eight months after the fourth antigen exposure to SARS-CoV-2, and t 1 = four weeks after the vaccination.We measured IgG antibody levels targeting the SARS-CoV-2 spike receptor binding domain (S-RBD-ab), potentially neutralizing antibodies by surrogate virus neutralization test (SVNT) against wild-type (Wuhan Hu-1), Delta (B.1.617.2),Omicron (B.1.1.529),and neutralizing antibody titers specific for BA.5 measured with a plaque reduction neutralization test (PRNT).During the six months following this vaccination, SARS-CoV-2 infections and symptom severity according to the World Health Organization guidelines were documented[13].In all centers, filtering face piece (FFP2) masks were still compulsory for patients and staff.

Figure 3 .
Figure 3. Omicron BA.5-PRNT titers before and four weeks after the vaccination.Percentage of study population grouped by SARS-CoV-2 Omicron BA.5-specific neutralizing antibody titer assessed using PRNT at two time points (t 0 = before and t 1 = four weeks after the vaccination with bivalent mRNA vaccine).(A) General view.(B) Spread in the group with the titers ≥1:160.Abbreviations: PRNT, plaque reduction neutralization test, **** (p ≤ 0.0001, Friedman test).

Table 2 .
Overview of used vaccines and antigen contacts.

Table 3 .
Differences in humoral immunity parameters and clinical features between the Omicron BA.5-PRNT positive and negative patients after vaccination with bivalent mRNA vaccine.

Table 4 .
Spearman's rank correlation between S-RBD-ab level and neutralization titers for wild-type, Delta, and Omicron variants of SARS-CoV-2 in surrogate virus neutralization test and Omicron BA.5-PRNT.