Development of Combined Human Papillomavirus and Rubella Vaccine

Ph.D Thesis Submitted by

Asmaa Mahmoud Ibrahim Gohar Master degree of Pharmaceutical Sciences – Cairo University (2016) Quality manager at Biotechnology unit National Organization for Research and Control of Biologicals (NORCB)

Submitted in Fulfillment for the Degree of Doctor of Philosophy in Pharmaceutical Sciences (Microbiology and Immunology)

Under The Supervision of

Professor. Dr. Magdy Ali Amin Professor of Microbiology and Immunology Faculty of Pharmacy, Cairo University

Dr. Nahla Shehata Dr. Nourtan F. Abdeltawab Assistant Professor of Biochemistry Assistant Professor of National Organization of Research and Microbiology and Immunology Control of Biologicals Faculty of Pharmacy, Cairo University

Department of Microbiology and Immunology Faculty of Pharmacy Cairo University 2019 Abstract

1 Abstract ABSTRACT Rubella vaccine was not part of the national immunization programs (NIP) in several countries in the Middle East and North Africa (MENA), South-East Asia (SEA), and South Africa regions until the year 2000. Therefore, immunization coverage of females older than 20 years old in these countries has been the focus of national campaigns for rubella elimination. Vaccines against human papillomavirus (HPV) are not part of NIPs in developing countries. To enhance the advantages of rubella-directed immunization campaigns and to increase HPV vaccine uptake in developing countries, this study aimed to test the stability, potency, efficacy and safety of a combined rubella and HPV vaccine. Female BALB/c mice were immunized subcutaneously with proposed combined HPV16 / HPV18 VLP and rubella vaccine at weeks (W) 0, 3 then with HPV vaccine at W 7. Immunized mice developed antigen specific antibodies against rubella and HPV significantly higher than mice immunized with rubella or HPV vaccine alone. The combined vaccine induced significantly higher splenocyte proliferation than control groups. In addition, pro-inflammatory cytokines IL-4, IL-6, IL-2, and IFNγ levels were significantly higher in mice immunized with the combined vaccine than control groups. Overall, the combined vaccine was safe and immunogenic offering antibody protection as well as eliciting a cellular immune response against rubella and HPV viruses in a single vaccine. This combined vaccine can be of great value to females above 20 years old in the SEA, MENA and South Africa regions offering coverage to rubella vaccine and a potential increase in HPV vaccine uptake rates after appropriate clinical testing.

2 Introduction

I. Introduction

Women in developing countries experience a shortage in the basic healthcare thus may face life-threatening health issues. Among the main health issues facing women in developing countries is maternal and infant mortality, human immunodeficiency virus (HIV) infection, breast, and cervical cancers (Nour 2008). Rubella causes miscarriage, premature delivery, and congenital rubella syndrome (CRS) that can cause fetal retinopathy, cataract, microphthalmia, hearing loss, congenital heart disease and hepatomegaly (Garcia et al. 2015). Chances of the fetus being affected by rubella are dependent on whether the pregnant mother was infected with rubella before or after conception (Duszak 2009). Rubella vaccine was Food and Drug Administration (FDA) approved in 1969 (Banatvala and Brown 2004) and was introduced as part of combined measles-mumps-rubella (MMR) vaccine in developed countries general immunization programs. However, in most developing countries rubella vaccine was not included in national immunization programs (NIP) until 2000. So, females older than 19 years old in these countries were not immunized with rubella vaccine that may threat their health and their prospective infants (Orenstein et al. 2018).

Egypt as a developing country and one of the most populous member countries of the World Health Organization (WHO) Eastern Mediterranean Region can be considered a model country for studying vaccine implementation. Egypt has achieved significant progress in vaccine preventable disease (VPD) control as it received technical and financial support from Centers for Disease Control and Prevention (CDC) and WHO. It is one of the developing countries with WHO regional office and following surveillance guidelines leading to current statistics on vaccine coverage and uptake rates.

3 Introduction

Egypt’s NIP is one of the most successful programs in the region with national immunization campaigns reaching > 95% coverage of the population. Rubella vaccine was introduced to Egyptian NIP in 1999 therefore; females older than 20 years old have not received rubella vaccine (El Sayed et al. 2011). In 2002, Egypt established a goal to eliminate measles and rubella and to prevent CRS by 2010, but large-scale rubella and measles outbreaks occurred in 2005–2007 made it difficult for Egypt to achieve the 2010 goals. The Egyptian Ministry of Health (MOH) made a strategy to eliminate rubella and measles by conducting a national catch up immunization campaign in 2008–2009 targeting 36 million children, adolescents and young adults from 2 to 19 years old (Gadallah et al. 2014), but females ≥ 20 years old were not included in this campaign and women in child bearing period who were born before 1999 did not get the compulsory vaccine.

Cervical cancer is one of the most common cancers among women worldwide and is highly associated with human papillomavirus (HPV) types 16 and 18 (Serrano et al. 2018). The majority of cervical cancers occur in women in the developing countries, where the accessibility and availabilities of vaccines and preventative screenings such as pap smears are limited (Demment et al. 2015). FDA approved HPV vaccines including: quadrivalent (HPV types 6, 11, 16, and 18), nonavalent (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58), and bivalent (HPV types 16 and 18) vaccines known by trade names Gardasil, Gardasil9 and cervarix, respectively (Schiller et al. 2012). FDA approved HPV immunization in males and females aged 9 through 45 years with continuous research on increasing the vaccination rates (Miller et al. 2018). Gardasil9 is indicated in males for prevention of anal cancers caused by HPV types 16, 18, 31, 33, 45, 52, and 58 and for prevention of genital warts caused by HPV types 6 and 11. It is also used to prevent anal intraepithelial neoplasia grades 1, 2 and 3.

4 Introduction Gardasil9 also showed increased impact compared to the quadrivalent vaccine in preventing High Grade Squamous Intraepithelial lesions (HSIL) (Capra et al. 2017). In addition, cervarix was shown to have potential to reduce the incidence of grade 3 or greater cervical intraepithelial neoplasia and cervical cancer (Lehtinen et al. 2012). In developing countries, HPV vaccine is not part of the routine immunization programs and is regarded as a costly vaccination. Over 25 million women over 15 years old are at risk of developing cervical cancer in Egypt. Moreover, females over 20 years old were not immunized against rubella, thus a combination between rubella and HPV vaccines could offer value to increasing vaccination rates of HPV while protecting against anogenital cancers and rubella teratogenicity.

Combination vaccines are considered as one of the solutions to minimize the vaccination cost and increase compliance while offering the same protection as individual vaccines with several successful combination vaccines as diphtheria, tetanus and pertussis (DTP) and MMR. HPV vaccine is a valuable preventive measure of anogenital cancers, thus, investing in a combined vaccine of HPV with an already successful NIP vaccine, rubella vaccine, can prove valuable in promoting vaccine uptake. Accordingly, protection against cervical cancers with a relatively low public health expenditure compared to cancer treatment expenses. Thus, the aim of this study was to combine rubella and HPV vaccines into a single vaccine that is safe, cost effective and potent.

5 Aim of work

II. Aim of work

1. Preparation of combined HPV and rubella vaccines

2. Evaluation of the safety profile of the combined vaccine through abnormal toxicity

test and serum biochemical parameters assay

3. Evaluation of the humoral and cellular immunity of the combined vaccine

4. Evaluation of the potency and stability of the combined vaccine

6 Review of Literature

III. REVIEW OF LITERATURE

3.1 Rubella

Congenital rubella syndrome (CRS) was discovered in 1940s in the United States after the ophthalmologist Norman Gregg linked the epidemic congenital cataracts to intrauterine rubella infection (Lambert 2007). At first, the only identifying characteristics of congenital rubella were cataracts, deafness, and congenital heart diseases. CRS causes also pathology in the brain, lungs, liver, spleen, kidney, bone marrow, bones and endocrine organs. It also causes pneumonia, hepatitis, thrombocytopenia, diabetes mellitus, and thyroiditis. There are other manifestations that occur in the eye, including glaucoma (Plotkin 2006).

3.1.1 Structure and classification of rubella virus

Rubella virus is a fragile virus, which is easily destroyed by detergent, heat and extremes of pH. It is considered as a member of the family Togaviridiae, but a sole member of the genus Rubivirus. Rubella virus virions particles are about 57 to 86 nm in diameter, positive sense RNA of 9762 nucleotides. Rubella virus contains three structural proteins, and two non- structural proteins. The three structural proteins are the capsid phosphoprotein, glycoprotein E1 and E2. The two non-structural proteins are P90 and P150 and they are involved in viral replication (Figure 1). The capsid protein interacts with the RNA genome and forms the nucleocapsid, which is surrounded by a lipid membrane upon which E1 and E2 are arranged (Chen and Icenogle 2006).

7 Review of Literature

3.1.2 Pathogenesis of acquired rubella infection

Rubella is most likely transmitted by direct or aerosol contact with upper respiratory tract secretions of infected people. Infectivity occurs one week before to two weeks after rash onset. After exposure, the virus attaches to and invades respiratory epithelium and then spread hematogenously to regional lymphatics. Primary viremia occurs five to seven days after infection. Virus replicates in local and distant reticuloendothelial sites and is followed by secondary viremia, especially during the second week of infection.

3.1.3 Immune response in acquired rubella infection and vaccine induced immunity

Humoral immunity to natural infection and rubella vaccine induced immunity is characterized by development of a variety of antibody types reactive to distinct viral antigens, including viral hemagglutinins, complement-fixing (CF) antigens, and precipitating antigens. Development of hemagglutination inhibition and neutralizing antibodies occurs with the onset of the rash illness, and CF antibodies are detected one week later. Peak titers of hemagglutination inhibition and neutralizing antibodies occur about two weeks after rash onset, but CF antibodies peak about two weeks later. The hemagglutination inhibition and neutralizing antibodies persist for life. The immunity that is induced due to natural or vaccine induced immunity is long lasting.

3.1.4 Epidemiology

Rubella is an acute viral disease that affects children and adults and distributed worldwide. Rubella is spread via the respiratory route from person to person.

8 Review of Literature

It causes congenital rubella syndrome that affects 110,000 children around the world every year (Hesarur et al. 2018). In Egypt, the burden of rubella was underappreciated until 2002. In 2002 and 2003, 274 and 261 confirmed rubella cases were reported, respectively, of which 45% occurred among children 5-9 years of age. In 2004, only 21 cases of rubella were reported. In 2005, 520 rubella cases were reported, of which > 80% among persons 11-20 years old. In 2006, 2587 cases were reported, almost 60% of which occurred among 11 to 20 year old patients. In 2007, the epidemic peaked with 11,345 cases reported, 74% of which were among 11 to 20 years old from 22 governorates. In 2008, the epidemic declined as 1,684 cases were only reported from 26 governorates. Following the 2008–2009 measles and rubella (MR) vaccination campaign, only 14 cases of rubella were reported in 2009, and by December 2010, only 15 confirmed cases of rubella have been reported

3.1.5 Rubella vaccines

There were several attempts towards the development of inactivated rubella virus vaccine, but these attempts were thwarted as the developed vaccine was not antigenic and no antibodies were produced. Also, several trials were performed to develop live attenuated virus vaccine. In 1965, Parkman et al. were the first to successfully attenuate the rubella virus with viral passage 77 times in African green monkey kidney. In 1969–1970, three vaccines were licensed in the U.S, including HPV-77 (dog kidney), HPV-77 (duck embryo) and Cendehill (rabbit kidney). All three vaccines were replaced by RA 27/3, which was licensed in Europe in 1970, as HPV-77 vaccines had several side effects, including arthritis, arthralgia and neuropathy. RA 27/3 is the most commonly used vaccine strain globally that was attenuated by passaging in human diploid fibroblast (WI-38), at temperature of 30ºC and 35ºC (Petrova et al. 2016).

9 Review of Literature

Due to the deficiencies of currently used rubella vaccine and the drawbacks associated with live attenuated vaccines, the development of alternative non- replicating, more improved types of vaccine are needed. Many studies have shown that viral protection is mostly induced by neutralizing anti-E1 antibodies (Hofmann et al. 2000). For these reasons, it has been postulated that the E1 glycoprotein might constitute a vaccine offering protection against rubella (Perrenoud et al. 2004). Perrenoud and others (2004) have shown that immunization of mice with this recombinant rE1 glycoprotein resulted in the production of an antibody neutralizing rubella virus, which is similar to naturally acquired rubella virus infection. E1 glycoprotein also contains epitopes needed for both T-cell, and B-cell, dependent immune response. Therefore, recombinant glycoprotein E1 may be used for candidate rubella vaccine production, and recent studies have suggested that it may be feasible for clinical trials (Perrenoud et al. 2004). Rubella virus antigenic determinant production in plants may become a promising way for development of candidate vaccine because plants and human have no common pathogens. Skryabin and his group (2009) have created viral expression vectors by modifying rubella virus protein E1 and agroinfiltrating them into Nicotiana benthamiana plants. The authors have suggested that recombinant protein E1 that is expressed in plants, might be used for rubella virus vaccine development (Petrova et al. 2016). The major drawbacks of using recombinant proteins are the small amounts that are obtained, and the high cost of purification. It has been also found in a number of studies that the expression of the three structural proteins of rubella virus (proteins C, E1, E2) in stably transfected CHO or BHK cell line resulted in non-infectious RVLPs production (Giessauf et al. 2005). Immunization of mice with RVLPs induced specific antibody responses against rubella virus structural proteins as well as

10 Review of Literature virus-neutralizing and hemagglutination-inhibiting antibodies (Qiu et al. 1994).

3.2 Human papillomavirus

Papillomaviruses (PV) are highly species-specific. PV causes epithelial infections in most mammals and birds. Until the end of 1960, there was not enough reproducible data that link cervical cancer to sexually transmitted diseases. In 1966, Francis Peyton Rous found that cottontail rabbit papillomavirus can induce malignant transformation and neoplastic growth in the skin of the rabbit. He concluded from his study that the virus can cause cancer in mammals. In the early 1970s, there were several studies that indicated the role of human papillomaviruses (HPV) in cancers as reviewed in (Zur Hausen 2009).

3.2.1 Structure and classification of papillomaviruses

HPV is a circular, non-enveloped and double stranded DNA virus. The virus DNA contains approximately 8000 base-pairs encoding eight genes. These genes are classified as early region (E) and late region (L) (Graham 2010). There are six E genes (E1, E2, E4, E5, E6 and E7) and two L genes (L1 and L2).

3.2.2 HPV prevalence and epidemiology

3.2.2.1 Burden of cancers attributable to HPV infection

HPV infection can cause cancer in the following sites: cervix uteri, anus, vulva, vagina, penis, oropharynex, oral cavity, larynex and other pharynx. HPV represented 29.5% of infection- related cancers worldwide in 2012. It causes approximately 630,000 new HPV related cancer cases per year, 570,000 of which occur in women and 60,000 in men.

11 Review of Literature

3.2.2.2 Burden of cervical cancer according to geographical distribution

Cervical cancer is most common in the developing countries, but represents less than 1% of all cancers in the developed countries. It the fourth leading cause of cancer deaths worldwide and the second in women aged 15- 44 years, with an estimated 528,000 new cases and 266,000 new deaths in 2012

3.2.2.3 Burden of other anogenital cancers:

HPV is responsible for many cancers, including the vulva, vagina, penis and anus. Globally, 115,000 cases of anogenital cancers are diagnosed, but only 68,500 cases are due to HPV infections. In men, nearly 30,000 cases are diagnosed with anus and penis cancers, but in women 38,500 cases are diagnosed with anus, vulva and vaginal cancers.

3.2.2.4 Burden of head and neck cancers

Head and neck cancers represent a major health problem worldwide. These cancers are more common in the developed countries. Head and neck cancers occur mainly due to alcohol and tobacco consumption. However, a fraction of these cancers are caused by HPV infection. HPV infection caused about 37,200 cases around the world from a total of 456,000 globally. It caused about 29,000 cases in the oropharynex, 4,400 in the oral cavity and 3,800 in the larynex in 2012.

3.2.2.5 Burden of genital warts

Genital warts are common manifestations of genital HPV infections. They are commonly associated with HPV6 and HPV11. However, many other HPV types have also been isolated from genital warts, including HPV 40, 42, 43 and 54.

12 Review of Literature

3.2.3 HPV vaccines

HPV vaccines are based on hollow virus-like particles (VLPS) assesmbled from recombinant HPV coat proteins. VLPS are not infectious, since they lack the virus's DNA. There are three available HPV vaccines: cervarix, gardasil and gardasil9. Gardasil is quadrivalent HPV (Types 6, 11, 16 and 18). It is used for prevention of both precancerous and cancerous conditions for lower genital tracts, anal area, head and neck. Cervarix is a HPV bivalent vaccine that protects from both HPV types 16 and 18. Regarding gardasil9, it was FDA approved in 2014. It is approved for the prevention of diseases like precancerous and cancerous condition in uterine cervix, vulva, vagina and cancer that caused by HPV types 16, 18, 31, 33, 45, 52 and 58. In addition, it protects against genital warts which are caused by HPV types 6 and 11. 3.3 Combination vaccines 3.3.1 Combination vaccine public health and economic values Vaccination is considered as one of the most cost effective public health measures and is effective in reducing morbidity and mortality. Over the few past decades, several vaccines were included in NIP schedule that made the vaccine implementation very difficult. The increase in the available vaccines not only impacted patients and parents compliance, but also affected the schedule complexity. Combination vaccines represent an important solution to the problem of increased numbers of injections during single clinic visit. Combination vaccines have several advantages including; reducing the administration cost; increasing the compliance rate due to fewer injections; reducing trauma to infants and simplifying the immunization schedule.

13 Summary

VIII. SUMMARY

The present work aimed to combine rubella and HPV vaccines into a single vaccine that is safe, cost effective and potent. The safety profile, immunogenicity, potency and stability of the proposed combined vaccine were evaluated in murine model. All quality control testing that were performed on the candidate vaccine were in compliance with Note for guidance on preclinical pharmacological and toxicological testing of vaccines according to EMA and WHO guidelines on non- clinical evaluation of vaccines. To examine the humoral response to combined vaccine, female BALB/C mice were divided to four groups receiving vaccines via subcutaneous injection. First group of mice were immunized with rubella vaccine at a dose of 3 log10 at week 0 and 4. The second groups of mice were immunized with HPV bivalent vaccine with a dose 2 μg/ mL at weeks 0, 3 and 7. For group three, mice were immunized with combined HPV- rubella vaccine at weeks 0, 3, then at week 7 mice were immunized with HPV alone. For group four, mice were injected with saline and served as a control group. Blood samples were collected from mice two weeks after each immunization, and then at week 12 and 24. Results showed that specific antibodies against HPV 16 L1 VLP antigen differed significantly between mice immunized with the combined vaccine or those immunized with HPV vaccine alone at all-time intervals. These results suggested that two doses of the proposed combined vaccine may be sufficient in inducing humoral immunity instead of three doses. HPV 18 L1 VLP antibodies differed significantly between mice immunized with the combined vaccine or those immunized with HPV vaccine alone at all-time intervals. Combined vaccine also elicited in vivo specific IgG antibodies against rubella significantly higher than mice immunized with rubella vaccine alone.

14 Summary

The safety profile was also evaluated by performing the abnormal toxicity test and assessing selected relevant serum biochemical parameters. The abnormal toxicity test was performed according to European pharmacopoeia monograph 01/2008: 20609. The results showed that there were no significant changes in body weight, activity, or food consumption in the immunized group compared to the control group. For serum biochemical parameters, all mice results were within the physiological ranges.

To evaluate the potency of the proposed combined vaccine, in-vitro relative potency by immune assay was performed to determine the effect of the combination on the antigen content of HPV. Results showed that the validity criteria were met and HPV antigen content was not affected by the combination. Concerning the stability study of the combined vaccine, the thermostability was evaluated by incubating the vaccine at 37ºC for seven days. The procedure for determining the thermostability was performed according to WHO TRS No.840 and results indicated that the vaccine was thermostable at the recommended incubation temperature. To evaluate immunogenicity of the combined vaccine, cytokines responses including, IFN- γ, IL-2, IL-4 and IL-6 were measured. BALB/C mice (n = 4 mice per group) were immunized with HPV, or rubella vaccines or their combination. Blood sera were collected 72 h after the last immunization and sera concentrations were determined. Results indicated that the proposed combined vaccine elicited significant IFN-γ, IL-2, IL-4 and IL-6 concentrations when compared to the individual vaccines and control group. Splenocyte proliferation assay was used to determine splenocyte activation and the cell-mediated immune responses. To evaluate splenocyte proliferation, female BALB/C mice (n = 4 mice per group) were immunized on day 0 with combined vaccine, and then at week 2 and 4, mice were immunized with HPV alone. Splenocytes were harvested 10 days after the last immunization. Results showed that splenocyte proliferation responses against the combined vaccine were significantly higher than negative control group.

15 Summary

Collectively, results showed that the proposed combined vaccine was safe in animal model, inducing significant antibody and cellular immunity. From the immungenicity results, the study suggested that two doses schedule may be used as alternative to three doses schedule of HPV. The dose reduction may add beneficial value in reducing cost and increasing compliance towards the proposed combined vaccine. The presented results support the interest of conducting clinical trials of the combined vaccine towards assessment of dosing and timing of administration of the proposed combined vaccine.

Conclusion 1. Combined vaccine elicited significant antibody production in comparison with the control group and the individual vaccine groups

2. Combined vaccine elicited significant cytokines production which play significant role in humoral and cell mediated immunity

3. Two doses schedule may be used as an alternative to three doses schedule of HPV

4. Combined vaccine was safe and demonstrated through abnormal toxicity test and serum biochemical analysis

5. The potency of HPV and rubella vaccine were not affected by the combination

6. The proposed vaccine was stable according to the WHO stability program

7. The presented results support the interest of conducting clinical trials of the combined vaccine towards assessment of dosing and timing of administration of the proposed combined vaccine.