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Efficacy and Safety of Sinopharm Vaccine for SARS-CoV-2 and breakthrough infections in Iranian Patients with Hemoglobinopathies: A Preliminary Report

Background The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to high morbidity and mortality worldwide. Vaccination against SARS-CoV-2 is a leading strategy to change the course of the COVID-19 pandemic. Aims of the study Our aim was to investigate the efficacy and side effects of the Sinopharm vaccine in patients with hemoglobinopathies in Iran and the frequency of breakthrough infection after a full course of vaccination. Methods A multicenter cross-sectional study of 434 patients with hemoglobinopathies (303 β-thalassemia major, 118 β-thalassemia intermedia, and 13 sickle-thalassemia) were conducted from March to July 2021 in IRAN. All patients have received the first dose of the China Sinopharm vaccine and received the second dose of the vaccine 28 days apar Antibody testing Detection of immunity after vaccination was evaluated by commercial enzyme-linked immunosorbent assay (Pishtazteb ELISA commercial kit), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total neutralizing antibody (NAb). Results The mean age of patients was 35.0 ± 8.5 (from 18 to 70) years, and 55.6% were positive for the antibody. Overall, 48.2% of the studied population had at least one side effect after vaccination. The most frequent side effects were fever and chills, dizziness, and body pain. A total of 90 (20.7%) vaccinated patients developed breakthrough infections after two doses of Sinopharm vaccination. Disease severity was recorded, and it was classified as mild in 77.8%, moderate in 13.6%, and severe in 7.4% of patients. One 28-year-old woman with β-thalassemia major died eight days after diagnosing a breakthrough SARS-CoV-2 infection. Conclusion No safety concerns were identified in patients who received two doses of the Sinopharm vaccine. Its efficacy was not optimal due to the lack of effect on new variations of the virus. However, our data show that it seems to be protective against the severity of COVID-19 infection in patients with hemoglobinopathies. The frequency of breakthrough infections after two doses of Sinopharm vaccination supports the evolving dynamic of SARS-CoV-2 variants requiring special challenge since such infection may represent a risk for vulnerable patients.

Introduction. The coronavirus disease (COVID-19) is caused by a positive-stranded RNA virus called severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), belonging to the Coronaviridae family. In humans, the virus causes COVID-19, a disease characterized by fever, shortness of breath, and pneumonia, which can be fatal in vulnerable individuals. 1 The most severe cases of COVID-19 with admission to the Intensive Care Unit (ICU) are generally more frequent in males and the elderly, especially those with comorbidities such as diabetes mellitus, obesity, chronic cardiovascular and/or respiratory disease. 2 The prevalence and mortality rates of SARSCOV-2 are changing on a daily basis2. According to World Health Organization (WHO), as of October 2021, the COVID-19 pandemic involved 216 countries and had affected 240,260,449 people, and that caused the death of 4,890,424. 3 Therefore, a prophylactic approach is crucial to control the disease, although the specific management against COVID-19 is still under investigation. To date, multiple vaccines have been developed worldwide utilizing different technologies, including messenger ribonucleic acid (mRNA) vaccines and classic inactivated virus vaccines. Each vaccine exhibits a different potency and duration of efficacy, as determined by the antigen design, adjuvant molecules, vaccine delivery platforms, and immunization method. Largescale clinical studies found that COVID-19 vaccines prevented most people from getting COVID-19 illness, but like most other vaccines, they are not 100% effective. [4][5][6][7][8] Sinopharm Beijing and Wuhan Institute of Biological Products have produced two inactive SARS-CoV-2 virus vaccines (CoronaVac vaccine developed by Sinovac and Sinopharm BBIBP-CorV) using chemical β-propiolactone. Both were treated with aluminumbased adjuvant to increase immunogenicity. A schedule of two doses (0/21-28-days) of the vaccine that can be stored at 2°C -8°C is recommended to prevent SARS-CoV-2 infection. 9 According to China National Biotec Group Company study, more than 40,000 people in the United The Arab Emirates and Bahrain, aged 18 and above without a known history of COVID-19, participated in the trials. The vaccine showed an efficacy rate of 79% against symptomatic COVID-19 cases and reported serious adverse events (AEs) after vaccination were rare. 10,11 On May 7, 2021, the World Health Organization (WHO) approved the vaccine, and it is used in many countries, including Iran, but the European Medicines Agency (EMA) has not yet reviewed its use for the European Union.
In Iran, a country with a population of around 85,451,701, COVID-19 affected 5,987,814 people and 127,299 mortalities were reported up to Nov. 8, 2021. In patients with hemoglobinopathies (thalassemias and sickle cell disease), several factors (iron overload, frequent hospital visits and admissions, immunodeficiency related to the disease itself, continuous use of medications, or associated complications) could predispose subjects to an increased risk for acquiring COVID-19 and consequent complications. [12][13][14][15] Moreover, higher mortality has been reported in subjects with thalassemias than the general infected population. 12 For all these reasons, patients with hemoglobinopathies were registered as the first group to get the vaccine in Iran.
We undertook this study to determine the efficacy and side effects of the Sinopharm vaccine in patient with hemoglobinopathies in Iran, and the frequency of breakthrough infection after two doses of Sinopharm vaccination.
Patients' data. All patients were diagnosed based on their clinical and laboratory data (clinical history, complete blood count and hemoglobin electrophoresis). Patients with transfusion-dependent thalassemia (TDT) were those requiring regular lifelong blood transfusions, starting before the age of 2 years with a hemoglobin level below 7 g/dL. Patients with non-transfusion-dependent thalassemia (NTDT) and sickle-thalassemia were Transfusions free or receiving occasional blood transfusions for a very limited period, such as surgery. All patients received two doses of Sinopharm vaccination. In subjects with a previous history of SARS-CoV-2 infection, the first Sinopharm dose was given three months after COVID-19. The interval between blood transfusion and vaccine injection was three days at least.
Patients under 18 years old, pregnant women, patients with fever and acute phase of the COVID-19 disease have not been included in the present report.
A designed questionnaire was made to collect all demographic, clinical, and laboratory data, including age, sex, age of diagnosis of a hemoglobinopathy, type of thalassemia, blood transfusion requirement (type and interval of blood transfusion), splenectomy status, serum ferritin level, preexisting comorbidities (diabetes, chronic heart failure, hypothyroidism, hypoparathyroidism, hypogonadism, cirrhosis, HCV positivity, osteoporosis, extramedullary hematopoiesis, thrombosis, liver, kidney and cardiac diseases, and pulmonary hypertension). In addition, a serum ferritin > 2,500 ng/mL was considered an indirect index of severe iron overload.
Patients' outcome and AEs after vaccination were recorded via interview or after reviewing patients' personal histories.
Breakthrough SARS-CoV-2 infection. "A breakthrough infection" was suspected in presence of clinical defined signs and diagnosed by detection of SARS-CoV-2 on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay, two weeks or more after the second dose of Sinopharm vaccine 8 and were classified into asymptomatic/mild, moderate, and severe, according to WHO. 16 Antibody testing. Detection of immunity after vaccination was evaluated by commercial enzymelinked immunosorbent assay (Pishtazteb ELISA commercial kit), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total and neutralizing antibody (NAb). The assays were performed according to the manufacturer's protocol. Elisa assay has a sensitivity and specificity of 94.1 % and 98.3%, respectively. A negative result can occur if the quantity of antibodies for the SARS-CoV-2 virus present in the specimen is below the assay's detection limit, or the virus has undergone minor amino acid mutation(s) in the epitope recognized by the antibody utilized in the test.
The tests were performed one month after the second dose of Sinopharm vaccination. 17 Statistical analysis. Data were analyzed by IBM SPSS version 23. Normality of data was checked by Kolmogorov-Smirnov test. Descriptive data were presented as mean, standard deviation, median, range, and percentage. Inferential analysis was performed by Student t-test or Mann-Whitney test to compare quantitative data between the two groups. Comparison of qualitative variables among different groups was made by the Chi-square test. A P-value less than 0.05 was considered statistically significant. The severity of breakthrough coronavirus infection in 81 out of 90 patients (90%) was classified as mild in 77.8%, moderate in 13.6%, and severe in 7.4%. The most common mutations of TDT were IVS-I-II and IVS-I-V with the genotype of β 0/ β 0 , and the most common mutations of NTDT were -101 and -92 with β + /β + genotype without co-inherited α-thalassemia. The mean pre-transfusion hemoglobin levels in TDT and hemoglobin levels in NTDT and sickle-thalassemia were 9.9 ± 1.1 g/dL and 9.7 ± 1 g/dL, respectively. We did not find any relationship of COVID-19 severity disease with the severity of thalassemia itself (P=0.717), hemoglobin levels (P=0.956), and patients' genetic mutations.
A 28-year-old female with β-thalassemia major (blood group O + , regularly transfused with washed packed red blood cells) presented with fever, dry cough, and dyspnea 120 days after the second dose of the Sinopharm vaccine. She had been compliant with   On the 4 th day of admission, she developed a severe and persistent headache requiring a brain computed tomography (CT) scan. No relevant changes were documented; however, a second brain CT scan, carried out 12 hours later, showed an intraventricular and left frontal lobe parenchymal hemorrhage with midline shift and herniation (Figure 1). Moreover, the CT venography detected a subarachnoid hemorrhage (Figure 2) without evidence of intracranial vascular thrombosis. Therefore, the patient underwent surgery due to impending herniation and midline shift. Unfortunately, a few hours after surgery, she developed hypotension and finally died. Coagulation assays (PT, INR, APTT, and platelet count) were normal prior to surgery, but PT and INR were prolonged after surgery (32.6 Sec, Control:13.8 Sec and 2.5, Reference range: 0.9-1, respectively), while PTT and platelet count were normal.
Chest CT scan showed diffuse bilateral lung involvement, typical for COVID-19 pneumonia. (Figure  3). The patient had no family history of bleeding tendency and was not receiving hormone replacement   therapy with oral estrogen/progesterone. Table 3 shows the association of some clinical and laboratory parameters related to the antibody status of vaccinated patients. They were divided into two groups based on positive or negative antibodies. The results revealed a significantly lower incidence of breakthrough coronavirus infection after full vaccination in the positive antibody group (8.2%) compared to the negative group (22.1%; P = 0.02). The other evaluated factors were comparable between the two groups (P= ˃0.05).
Moreover, we evaluated the laboratory, and clinical features of post vaccinated patients with and without Sars-CoV-2 infection ( Table 4). Only one parameter resulted statistically significant: the median of serum ferritin level in patients who developed a breakthrough coronavirus infection ( Table 4; P = 0.013).

Discussion.
Since there is no definitive therapy for COVID-19, vaccination is the only appropriate approach for controlling the disease. So, vaccines are a critical tool in managing the COVID-19 pandemic.
According to the type of vaccine, variable immune responses and side effects are seen. [6][7][8] Several vaccines have been rapidly developed and approved by many countries for emergency use.
One of these vaccines is Sinopharm (Beijing Institute of Biological Products Co., Ltd, China) that is an inactive SARS-CoV-2 virus vaccine. 12 An interim recommendation for its use has been reported by the  A cross-sectional study of 1,102 attendants (aged ≥18 years) in the UAE who received two doses of Sinopharm vaccine showed that, in general, it was tolerated, and post-vaccination AEs were mild without any serious reported adverse event. 10,11,24 Our data confirm these observations in our patients with hemoglobinopathies too.
Based on antibody assessment, the reported efficacy rate of the Sinopharm vaccine is between 73% -86% 11,23 , but our preliminary data have shown a lower level of immunity or protection from COVID-19 (55%) after two doses of vaccine with 28 days apart. Twenty-one of vaccinated patients developed breakthrough coronavirus infection at least one month after the second dose of Sinopharm vaccine. Although in the vast majority of patients with hemoglobinopathies, who developed post-vaccine COVID-19 disease, the symptomatology was mild-moderate, in 7.4% of them, the disease was severe, and one female patient died, three months after the second dose of vaccine, for a breakthrough coronavirus infection. Therefore, the efficacy of Sinopharm against SARS-CoV-2 infection (COVID-19) seems to be partial and less protective compared to other types of vaccines, as reported in the general population. [18][19][20][21][22][23] However, specific studies are needed to assess the immunogenicity, protective efficacy, and durability of immune response in subjects with hemoglobinopathies.
A breakthrough infection post coronavirus vaccine was also observed among the positive antibody testing group, although the incidence was significantly lower (8.2%) than the negative group (22.1%), reaffirming its suboptimal immunogenicity and protective effect in our patients with hemoglobinopathies. Since several variants of SARS-CoV-2 have been described during the pandemia, only a few are considered variants of concern (VOCs) by the WHO; it is pertinent to think that one of these variants may have decreased the vaccination effectiveness.
SARS-CoV-2, like other RNA viruses, is prone to Pag. 7 / 8 genetic evolution while adapting to their new human hosts with the development of mutations over time, resulting in the emergence of multiple variants that may have different characteristics compared to its ancestral strains. These adaptive mutations in the viral genome can alter the virus's pathogenic potential, and even a single amino acid exchange can drastically affect a virus's ability to evade the immune system. 25,26 One more interesting finding documented in our patients was the significantly higher median of serum ferritin level in the group of subjects with breakthrough coronavirus infection, supporting the potential role of iron overload on the immune system that may predispose patients to severe iron overload to SARS-CoV-2 infection (Table 4). [12][13][14][15] Therefore, the impact of iron overload and the benefit of strict adherence to iron chelation therapy should be recommended in these vulnerable patients in association to close heart monitoring, as reported in a previous study showing that heart failure and pulmonary hypertension are significant risk factors for COVID-19 severity in thalassemia patients. 27 Nevertheless, a 28-year-old β-thalassemia major woman, with no significant past medical history of comorbidities or bleeding tendency, died during breakthrough coronavirus disease while she got the second dose of vaccine four months prior to COVID-19 infection. Hypercoagulability is a known complication of COVID-19. Although severe cases are usually reported in older populations and in those with underlying comorbidities, our case emphasizes that this may occur in young without significant medical history. Intracerebral hemorrhage (ICH) was reported in a 5-case series with COVID-19 without having an underlying vascular abnormality, as we observed in our case.
All cases were on anticoagulants, and the time between symptom onset and ICH identification was 14 to 38 days in this case series, but it was shorter in our case (one week after starting COVID-19 symptoms). 28 The exact mechanism of COVID-19 associated ICH is not obvious at this young age case series , and our case supports the hypothesis that endothelial toxicity and disruption of the renin-angiotensin system might play a role in COVID-19-mediated ICH. [28][29][30] The recent emergence of multiple variants of SARS-CoV-2 has become a significant concern for public health worldwide. New variants have been classified either as VOCs or variants of interest (VOIs) by the CDC (USA) and WHO. VOCs are associated with enhanced transmissibility or virulence, reduction in neutralization by antibodies obtained through natural infection or vaccination, the ability to evade detection, or a decrease in therapeutics or vaccination effectiveness. The common feature of these variants is that they share the N501Y mutation involving the SARS-CoV-2 spike (S) protein, which is precisely the target of most COVID-19 vaccines. Furthermore, mutations such as N501Y, E484K, and K417N in the S protein may affect viral fitness and transmissibility. However, current research on the impact of these variants on COVID-19 vaccines is still lacking. 31 SARS-CoV-2 and its variants continue to cause great damages across the world. We advise that even fully vaccinated people continue to follow all safety precautions. Since the beginning of the pandemic, a preventive infection program has been recommended by the Iranian Ministry of Health that includes social distancing, wearing a mask, ward hospital isolation of infected patients during blood transfusion, and hand washing. Continued viral surveillance of new variants must be performed at regular intervals with viral genomic sequencing, given the possibility that more highly transmissible, more virulent variants and treatment-resistant variants could emerge. 32 Moreover, getting a booster shot may help prevent a breakthrough infection or have symptoms, as recommended by the CDC. 33 Conclusions Although the safety concern of Astra Zeneca was evaluated in three patients with S/B 0 thalassemia, 34 the current research is the first study that has evaluated the safety and efficacy of the COVID-19 vaccine in a large population of patients with hemoglobinopathies. No safety concerns were identified in our patients who received two doses of the Sinopharm vaccine. However, its efficacy is not optimal and is associated with a relatively low level of antibodies against COVID-19 (55%), which indicates a non-full protective effect. However, it is well-tolerated and seems to reduce the risk of severe breakthrough COVID-19 infection among patients with hemoglobinopathies. The frequency of breakthrough infections after full Sinopharm vaccination supports the evolving dynamic of SARS-CoV-2 variants requiring special challenge since such infection could represent a risk for vulnerable patients.