**7.1 Early age when immature immune system of neonates**

The most noteworthy risk group for extreme RSV infection is infants under a half year of age [78, 79]. Practically speaking, first dose should be administered at the age of 2 months. Full term newborn children obtain maternal antibodies during the latter 50% of gestation and levels of antibody remain moderately high for a half year after the birth [80]. This would interfere when RSV vaccine would be done [81]. So there is need of an ideal vaccine which will not interfere with the maternal antibodies and will give protection in the presence of maternal antibodies. A few investigations show that newborn children under the age of 8 months have a less serum counter acting agent (antibody) response to characteristic RSV disease as compared to elder ones [82]. A less developed immune system may be the reason of this reduced immunity level, but maternal antibodies may also suppress the immune response [83].

Recent schedule for hepatitis B, diphtheria, rotavirus, pneumococcus, pertussis, tetanus, *Haemophilus influenzae* type b and poliovirus show that vaccine for these infectious diseases will be done ideally after birth at 2, 4 and 6 months of age. Vaccination for RSV should be ideally administered at 6 months of age, so it is necessary and important to make sure that RSV vaccine should not interfere the working and efficacy of other routinely used vaccines during the childhood [84].

## **7.2 Induction of low affinity neutralizing antibodies**

RSV vaccine was developed shortly after it was isolated. In 1960, FI-RSV vaccine was injected by intramuscular route in 2–7 months old infants and children. Instead of providing protection against wild type RSV infection, FI-RSV enhanced the respiratory disease development following wild type RSV infection during the subsequent RSV season. Lungs of children and infants with enhanced diseased were rich with large numbers of eosinophils and this was not found in patients of natural infection with RSV. After this disastrous outcome, there was need to develop a safe RSV vaccine including the evaluation of enhanced disease [47–49, 85, 86].

These different immunopathology aspects which were seen in humans after FI-RSV vaccine and enhanced disease were later studied in non-human primates. In newborn macaques which were FI-RSV vaccinated and then infected with RSV virus, enhanced disease with increased level of eosinophils and neutrophils were seen [87–91]. FI-RSV produced the increased level of ELISA titer RSV antibody because it was highly immunogenic, but the provoked antibodies were non-neutralizing. Antibodies produced did not provide the protection against virus because it could not prevent the fusion of virus [92, 93]. FI-RSV induced resulted RSV antibodies were also known to be of decreased avidity and this may be the result of having lack of maturation [94–97].

#### **7.3 Lack of appropriate animal models**

No ideal animal model for RSV vaccine is present which can be used for its evaluation. African green monkey kidney cells (Vero cells) were used for production of RSV. High titers of RSV were observed on Vero cell line. Similar results were obtained when grown on human cells (HEp-2). On both these cell lines, RSV infection led to syncytia formation. These cell lines were used extensively to characterize the live attenuated RSV vaccines. In recent studies, there are reports that NHBE

(normal human bronchial epithelial) and HAE (human airway epithelial) cells are used to create model human nasopharyngeal mucosa. RSV infection did not show any pathological sign and also not led to syncytia formation on NHBE and HAE cell lines [98, 99].

Experiments to study attenuation of live attenuated RSV vaccine were also conducted on BALB/c mice, found permissive to infection to some extent. Advantages for mouse studies are that reagents are readily available which can be used for measurement of infection immune correlates [99–104]. Several nonhuman primates act as host for RSV. RSV can replicate in the nasopharyngeal tract of their host. Macaques, African green monkeys, Chimpanzees and bonnet monkeys have been used to model RSV infection [105–112]. Relative viral titers of live attenuated RSV vaccines compared to wild type RSV disease can also be measured. Chimpanzees are the only non-human hosts which develop and show the clinical sign and symptoms of coryza following RSV infection. They are much permissive to RSV infection. So they are used for evaluation of comparative level of attenuation among vaccines which are candidates in humans. But it has been shown by the recent studies that chimpanzee is not completely predictive of attenuation in young newborns. They are also scare and expensive. Study conducted by Karron et al. showed that those RSV vaccines sensitive to temperature and also had high degree attenuation in chimpanzee, were able to produce infection in lower respiratory tract in children [112].

#### **7.4 Absence of RSV disease liability data and commercial risk**

It is known that those children and persons primed with RSV are not at risk to RSV enhanced illness, but the absence of enhanced disease illness in RSV primed persons does not support the prediction that it will not be present in RSV naïve population. So it is very difficult to build up safety data which can support and be used for testation of novel RSV vaccine in newborns having age <6 month which is primary target population [113, 114]. There is absence of information (data) on RSV related mortality. This has prevented exact appraisal of the expenses and advantages of RSV vaccines and prioritization of vaccines for various target populaces [49]. Lack of information on disease liability data is a big problem in less developed countries where mortality cases are concentrated [115].

#### **7.5 Limited resources**

Clinical investigations of applicant vaccines in the target populace are fundamental to figure out which vaccines ought to be created for licensure, yet these examinations are tedious and costly, what's more, assets for these investigations are restricted. Measurement of impact of vaccine on disease in all target populations is very difficult and problematic. It is easy to diagnose RSV infection in infants and children because their respiratory secretions have high titers of RSV and so are easy to detect. Titers of virus in adults are low and sensitive RT-PCR assay is used for detection purpose. If there is a decrease in severity of disease, it is a good indication of vaccine being effective. Measures done for disease severity are not accurate at all ages of target populations. So there is need that larger and most costly studies should be performed [116].

#### **7.6 Emerging RSV variants/mutation in RSV genome**

RSV is divided antigenically into two groups which are RSV-A and RSV-B. These groups are further divided into genotypes as well as variants. It has been

**85**

*Hurdles in Vaccine Development against Respiratory Syncytial Virus*

investigated that different viruses belong to these different groups; genotypes as well as variants co-circulate in epidemics. So it is very difficult to develop an effective vaccine due to the presence of virus antigenic diversity as well as variability.

Spontaneous type of deletions of G and SH genes have been studied *in vitro*. RSV genome encodes 11 proteins; one of them is G protein which is most variable having 2 hypervariable regions. G protein has been investigated to accumulate amino acid changes periodically. RSV genotypes having the amino acids duplications in G

Researches had shown that formalin inactivation caused the alterations in the epitopes of the G and F proteins and as a result non-neutralizing antibodies were developed which led to formation of immune complex in the lungs [57, 69]. Recent studies have shown that changes in the properties of F protein occur during interaction of virus and host cell. The pre fusion (pre-F) which is highly energetic, transitions irreversibly into post fusion (post-F) form which is low energetic and stable, this occurs during insertion process of virus into host cell membrane. By this process, fusion of virus to host occurs. Although pre and post F are not structurally similar, they share 2 antigenic regions. Neutralizing antibodies target these antigenic regions. Pre F also has 3 other antigenic sites not present in post-F. These sites are neutralization sensitive [68–72]. It is investigated that pre-F conformation changes to post-F conformation during the mechanism of formalin as well as heat inactivation and this change is irreversible. As a result of this change, complete loss of epitopes occurs. So this process explains the one of the reasons for failure of

**7.8 Older age when immune-senescence of the elderly people**

**8. Future horizons in RSV vaccine and RSV therapeutics**

subunit vaccines for this target group [115, 117].

Elder target population group possess a considerable disease burden. The elder

group has preexisting immunity, which makes it inconvenient to increase the existing immunity. Furthermore immune-senescence may lead to decrease in the efficacy of vaccine [116]. Immune-senescence is a challenge for proper vaccination in older target populations. RSV disease burden increases in elderly people in presence of underlying diseases such as cardiac and pulmonary conditions. Live attenuated vaccines are found not to be immunogenic in elderly people. So now focus is on

• Continuous struggles are going on for the development of effective and safe RSV vaccines for each target group (infants, children, elders including pregnant women). Previous struggles made to build up a safe vaccine were failed. High antibody production was seen by the use of FI-RSV vaccine in 1960. However, unfortunately vaccinated children developed a severe disease after administration of FI-RSV vaccine. Difficulties and barriers associated with vaccine development particularly live attenuated vaccine are enhanced respiratory disease, maternal antibodies, nasal congestion, low immunogenicity, genetic variability and instability, immature immune system of infants, vaccine virus transmission and immune-senescence as well as preexisting immunity in elders. However, these problems are being slowly overcome [118].

–10<sup>−</sup><sup>4</sup> ).

Like other RNA viruses, RSV has high nucleotide substitution rate (10<sup>−</sup><sup>3</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.87126*

proteins also have been isolated.

FI-RSV [69, 71].

**7.7 Disruption of antigenic epitopes**

#### *Hurdles in Vaccine Development against Respiratory Syncytial Virus DOI: http://dx.doi.org/10.5772/intechopen.87126*

investigated that different viruses belong to these different groups; genotypes as well as variants co-circulate in epidemics. So it is very difficult to develop an effective vaccine due to the presence of virus antigenic diversity as well as variability. Like other RNA viruses, RSV has high nucleotide substitution rate (10<sup>−</sup><sup>3</sup> –10<sup>−</sup><sup>4</sup> ). Spontaneous type of deletions of G and SH genes have been studied *in vitro*. RSV genome encodes 11 proteins; one of them is G protein which is most variable having 2 hypervariable regions. G protein has been investigated to accumulate amino acid changes periodically. RSV genotypes having the amino acids duplications in G proteins also have been isolated.

## **7.7 Disruption of antigenic epitopes**

*The Burden of Respiratory Syncytial Virus Infection in the Young*

lines [98, 99].

in children [112].

**7.5 Limited resources**

should be performed [116].

(normal human bronchial epithelial) and HAE (human airway epithelial) cells are used to create model human nasopharyngeal mucosa. RSV infection did not show any pathological sign and also not led to syncytia formation on NHBE and HAE cell

Experiments to study attenuation of live attenuated RSV vaccine were also conducted on BALB/c mice, found permissive to infection to some extent. Advantages for mouse studies are that reagents are readily available which can be used for measurement of infection immune correlates [99–104]. Several nonhuman primates act as host for RSV. RSV can replicate in the nasopharyngeal tract of their host. Macaques, African green monkeys, Chimpanzees and bonnet monkeys have been used to model RSV infection [105–112]. Relative viral titers of live attenuated RSV vaccines compared to wild type RSV disease can also be measured. Chimpanzees are the only non-human hosts which develop and show the clinical sign and symptoms of coryza following RSV infection. They are much permissive to RSV infection. So they are used for evaluation of comparative level of attenuation among vaccines which are candidates in humans. But it has been shown by the recent studies that chimpanzee is not completely predictive of attenuation in young newborns. They are also scare and expensive. Study conducted by Karron et al. showed that those RSV vaccines sensitive to temperature and also had high degree attenuation in chimpanzee, were able to produce infection in lower respiratory tract

**7.4 Absence of RSV disease liability data and commercial risk**

countries where mortality cases are concentrated [115].

**7.6 Emerging RSV variants/mutation in RSV genome**

It is known that those children and persons primed with RSV are not at risk to RSV enhanced illness, but the absence of enhanced disease illness in RSV primed persons does not support the prediction that it will not be present in RSV naïve population. So it is very difficult to build up safety data which can support and be used for testation of novel RSV vaccine in newborns having age <6 month which is primary target population [113, 114]. There is absence of information (data) on RSV related mortality. This has prevented exact appraisal of the expenses and advantages of RSV vaccines and prioritization of vaccines for various target populaces [49]. Lack of information on disease liability data is a big problem in less developed

Clinical investigations of applicant vaccines in the target populace are fundamental to figure out which vaccines ought to be created for licensure, yet these examinations are tedious and costly, what's more, assets for these investigations are restricted. Measurement of impact of vaccine on disease in all target populations is very difficult and problematic. It is easy to diagnose RSV infection in infants and children because their respiratory secretions have high titers of RSV and so are easy to detect. Titers of virus in adults are low and sensitive RT-PCR assay is used for detection purpose. If there is a decrease in severity of disease, it is a good indication of vaccine being effective. Measures done for disease severity are not accurate at all ages of target populations. So there is need that larger and most costly studies

RSV is divided antigenically into two groups which are RSV-A and RSV-B. These groups are further divided into genotypes as well as variants. It has been

**84**

Researches had shown that formalin inactivation caused the alterations in the epitopes of the G and F proteins and as a result non-neutralizing antibodies were developed which led to formation of immune complex in the lungs [57, 69]. Recent studies have shown that changes in the properties of F protein occur during interaction of virus and host cell. The pre fusion (pre-F) which is highly energetic, transitions irreversibly into post fusion (post-F) form which is low energetic and stable, this occurs during insertion process of virus into host cell membrane. By this process, fusion of virus to host occurs. Although pre and post F are not structurally similar, they share 2 antigenic regions. Neutralizing antibodies target these antigenic regions. Pre F also has 3 other antigenic sites not present in post-F. These sites are neutralization sensitive [68–72]. It is investigated that pre-F conformation changes to post-F conformation during the mechanism of formalin as well as heat inactivation and this change is irreversible. As a result of this change, complete loss of epitopes occurs. So this process explains the one of the reasons for failure of FI-RSV [69, 71].

### **7.8 Older age when immune-senescence of the elderly people**

Elder target population group possess a considerable disease burden. The elder group has preexisting immunity, which makes it inconvenient to increase the existing immunity. Furthermore immune-senescence may lead to decrease in the efficacy of vaccine [116]. Immune-senescence is a challenge for proper vaccination in older target populations. RSV disease burden increases in elderly people in presence of underlying diseases such as cardiac and pulmonary conditions. Live attenuated vaccines are found not to be immunogenic in elderly people. So now focus is on subunit vaccines for this target group [115, 117].
