VIRAL VECTORS

Abstract For many human pathogens the traditional vaccine development platforms are unsuitable Owing to safety concerns Poor efficacy & Impracticality The alternative is therefore recombinant viral vectors as a means of vaccination Can express foreign protein at high levels in host cells Resulting in strong, long lasting immune responses

Introduction As compared to killed or inactivated virus vaccines, live attenuated vaccines produce better results However for some human pathogens safety issues arise due to under attenuation and even reversion to its pathogenic state e.g HIV Live attenuated simian immunodeficiency virus (SIV) Un fortunately SIV can also cause AIDS in monkeys due to reversion to pathogenecity

Even licensed live attenuated vaccines are not without the risk of vaccine mediated disease Small pox vaccination Vaccinia virus was used This has significant side effects that can be even life threatening Some viruses e.g Ebola and Marburg viruses are so deadly that live attenuated vaccine is not even considered Even subunit and recombinant vaccine might contain post translational modifications after expression in host cells that might alter their antigenicity These can also be degraded by the recipient immune system

In case of DNA vaccines all the discussed problems are eliminated But Expression of DNA vaccines is notoriously weak in vivo, resulting in lower immunogenecity of the vaccine Most promising recombinant vaccine technology platform is viral vectors Idea is to present the naturally occurring forms of the target pathogen’s antigens to the immune system in the absence of the pathogen itself Similar to natural infection however in the absence of the disease 1984, vaccinia virus vector to express rabies virus glycoprotein (Raboral VR-G)

Most studied viral vectors as vaccine tools Adeno virus Adeno-associated viruses Alphaviruses Newcastle disease virus Poxviruses Vesicular stomatitis virus, etc

ADENOVIRUS Most widely studied viral vector for vaccine and gene therapy Family: Adenoviridae Double stranded DNA viruses with genome of approx: 36kbp Of the human adenoviruses there are at least 51 different serotypes Ad 2 and Ad 5 are most well studied in terms of viral vectors Genomes are easy to manipulate, can be grown and purified to high titers in cell cultures and are able to infect a wide variety of dividing and non dividing cell types Also have a favorable safety profile

ADENOVIRUS GENOME

It has been established that Ad genome can package upto 1 It has been established that Ad genome can package upto 1.8kb of exogenous DNA (transgene) Later it was found that additional space for transgene can be created by specific deletion of Ad genome The first of these deleted genes were E1a and E1b which produce a replication incompetent vector due to loss of E1 This replication incompetent vector can only replicate in permissive cell line HEK293 which provides the missing E1 gene function for Ad vector Additional deletions are made in E3 gene These E1/E3 deleted Ad vector provide 4-5kb space for transgenes (first generation Ad vectors) Efforts for low expression of Ad genes and high expression of gene inserts were made

This was achieved through deletions in the E2 region or the E4 region which reduced the expression of E2 and E4 genes These Ad vectors are referred to as second generation Ad vectors With increased transgene capacity of upto 6-7kb Ad vectors for the highest capacity for exogenous DNA are called “gutless” vectors Consist of solely the exogenous DNA flanked by Ad inverted terminal repeat (ITR) and the Ad packaging signal These vectors can accommodate 30-35kb of foreign DNA Although they must be propagated with a helper virus to provide the missing Ad genes necessary for replication in the packaging cell line

SUMMARY OF AVV CAPACITY Designation/ Name of the Vectors Capacity to carry transgene Adenovirus (full genome intact) 1.8kb First generation Adenovirus vectors (E1-E3 deleted Adenovirus) 4-5 kb Second generation Adenovirus vectors (E1-E3 and E2-E4 deleted Adenovirus) 6-7 kb Gutless Adenovirus (All genes deleted except ITR and packaging signals) 30-35 kb

Ad vectors have been studied on many fronts Ebola Dengue Marburg Avian influenza West Nile virus SARS-CoV HIV and Anthrax Also have been studied as gene therapy vectors for various cancers

Primary criticism is the issue of pre-existing immunity 35-55% of the Ad population has neutralizing Abs, in particular against Ad5 These Abs might limit Ad based vaccines vector’s efficacy Use of alternate serotypes that are antigenically different and thus cannot be neutralized by these Abs is an option in this respect However still conflicting data exists in this regard Incase of Merck Ad-5 based HIV vaccine, the effect of neutralizing Abs can be overcome by increasing the dose of the vaccine in clinical trials

Evidence also exist that alternative route of administration (oral or intranasal) instead of injection can overcome pre-existing vector immunity The issue of pre-existing Ad vector immunity is a source of frequent debate and experimental data exist for support on both sides of the argument.

Adeno-Associated Virus (AVV) Belong to family Parvoviridae Small single stranded DNA viruses with genome of ~ 5kb 8 known AVV serotypes, AAV 2 being the most commonly studied Unique in sense that they need a helper virus to replicate e.g Ad virus or Herpes virus In the absence of helper virus, infection becomes latent = no viral progeny Wild type AVV do not produce disease in humans Thus have excellent safety profile 16

Persistent transgene expression in host cells Advantages Broad host range Persistent transgene expression in host cells Generate very weak antivector immune responses Recombinant AVV have been studied as vaccine vectors against Herpes simplex virus 2 HPV HIV Cytomegalovirus 17

rAVV can only accommodate ~5kb of exogenous DNA This is done by deleting almost all of the vector genes between the 3’ and 5’ terminal repeat sequences Must be packaged in special cell lines that express AVV rep and Cap proteins In addition to a helper virus Advantage is that there is no expression of parent virus genes, resulting in low antivector immunity Disadvantage is that in absence of helper virus the viral genome gets integrated into the human genome at chromosome 19 18

Thus raises safety issues Related to genetic consequence of genome integration (both beneficial and detrimental) Pre-existing Immunity Over 90% of humans have circulating Abs that cross react with AVV And 30% are serotype positive for AVV neutralizing Abs Highest level of AVV Abs are against AVV2 serotype Alternate serotypes as vector backbone can be used On July 26th 2007 U.S FDA announced the death of a clinical trial participant involving an AVV based RA treatment Details of the tragedy have still not been released 19

AlphaVirus Belong to family Togaviridae Small enveloped viruses, with single stranded positive RNA genome of ~ 11.8kb Arthropod borne viruses(arbovirus) and are grouped into 6 clades (based on antigenic homology of E1 glycoprotein) Barmah Forest (BF) Ndumu (NDU) Semliki Forest (SF) Western equine encephalitis (WEE) Eastern equine encephalitis (EEE) Venezuelan equine encephalitis (VEE) 20

By deleting structural genes and replacing them with transgenes Have a broad host range and can infect a variety of cell types including dentritic cells (APCs) (can directly target DCs and produce strong immune responses) Primary method of using alphaviruses as vaccine vectors is to create REPLICONS By deleting structural genes and replacing them with transgenes The recombinant RNA must be co expressed in packaging cell line with helper RNA containing the missing structural genes Thus the replicons are enveloped viral particles containing the recombinant genome that when expressed in the cell line produces the transgene at high levels One pitfall is the recombination between helper RNA and recombinant RNA Which produces replication competent alphavirus particles 21

The genome capacity of alphaviruses is also low ~5kb These have been studied as vaccine vectors for avian influenza Marburg virus Ebola virus HIV SARS-CoV Anthrax & Botulinum toxin 22

Pre-existing Immunity VEE replicons containing HIV genes were tested in phase 1 clinical trials and were well tolerated in vaccine recipients Pre-existing Immunity Not as significant as in the case of Ad viruses and AAV Largely because these are zoonotic , mosquito borne viruses that are endemic only in some geographical regions of the world Also human alphavirus epidemics occur very in frequently However there is evidence that pre existing antibodies in horses against one alphavirus strain can interfere with infection of another strain 23

New Castle Disease Virus (NDV) Belongs to family Paramyxoviridae Zoonotic virus that infects all species of birds Nonsegmented, single stranded negative sense RNA of ~15kb Antigenically different from any of human paramyxoviruses Categorized into 3 groups The avirulent lentogenic strains Moderately pathogenic mesogenic strains Highly pathogenic velogenic strains 24

Have been studied against SARS-CoV Respiratory syncytial virus SIV Lentogenic strains are widely used for NDV live attenuated vaccines in the poultry industry NDV are nonpathogenic in primates and thus has lead to their study as vaccine vectors Have been studied against SARS-CoV Respiratory syncytial virus SIV Influenza virus Despite the danger of NDV in poultry, these viruses have a safe profile in humans Since NDV is an avian paramyxovirus, the issue of pre existing immunity is not considered Also are used in many veterinary vaccines and are well characterized 25

NDV vectors may be somewhat limited in their capacity for large transgene inserts Most recently Sendai virus vectors have been shown to accommodate 4.5 kb of exogenous DNA 26

Pox Viruses Belong to family Poxviridae Large double stranded DNA viruses Viron size 350 x 270nm Genome size 300kb Most virulent virus is the variola virus an obligate human pathogen that causes smallpox Other noteable viruses are Vaccinia virus Monkeypox virus Cowpox virus (zoonotic viruses) 27

Are zoonotic arboviruses that are nonpathogenic in humans Poxviruses of the genus Avipoxvirus have attained research attention as vaccine candidates Are zoonotic arboviruses that are nonpathogenic in humans Also have genome size of 260kb Fowlpox and canarypox have been tested in animal models as vaccine vectors for rabies H5N1 avian influenza Nipah virus HIV Since these are zoonotic viruses, pre existing immunity is not considered to be an issue Vaccinia viruses have been used as vaccines for decades 28

Large size can accommodate large transgenes, but In case of vaccinia virus, much of the adult human population today is seropositive for vaccinia due to childhood smallpox vaccination Therefore a vaccinia based vaccine vector in these individuals would be in effective due to antivector immunity The large genome size of poxvirus is both advantageous and disadvantageous Large size can accommodate large transgenes, but The expression of parent virus proteins can produce strong immune responses that lead to reduced vaccine efficacy This could be an explanation of poor performance of poxviruses in human clinical trials In direct comparison a recombinant Ad vector expressing a transgene induces much stronger cellular immune responses than a vaccinia virus expressing the same gene 29

Vesicular Stomatitus Virus (VSV) Belong to Rhabdoviridae (same family as rabies virus) Zoonotic arboviruses 11kb genome of single stranded negative sense RNA VSV transmission to animals takes place through insect bites Can cause severe disease in cattle, horses and swine with symptoms similar to foot and mouth disease In humans infections occur less frequently and also with less severe disease In form of mild flu like symptoms rVSV can accommodate a 40% increase in genome size with only a slight reduction in infectivity titer 30

Concerns for VSV vector safety are related to severe human disease Another advantage is that the virus can efficiently incorporate and express foreign transmembrane proteins on the surface of recombinant viral particles Concerns for VSV vector safety are related to severe human disease As well as neurovirulence and 50% mortility rate from experimental intranasal mouse infections Asymptomatic brain infections have also been noted in experimental models after intranasal delivery of VSV vector 31

rVSV vaccine vectors have still been studied against H5N1 Ebola and Marburg viruses Plague Hepatitis C virus HIV Pre existing immunity is not considered an issue in this case as well However in cases where it does exist, the option of serotype rotation is open, similar to Ad and AAV vaccine vectors 32

Other Viruses Herpes simplex virus (SIV, HIV and bacterial pathogens) Measles virus (HBV, HIV and West Nile Virus) Poliovirus (HBV and SIV) 33

Summary Viral vectors are suitable for presenting naturally formed antigens to the immune system Have more favorable safety profile than live attenuated vaccines Are more immunogenic than inactivated or killed virus vaccines Present the desired antigens in the correct conformation in comparison to subunit vaccines Express high levels of foreign genes in vivo than DNA vaccines 34

Considerations Safety (Ad vectors considered generally safe but VSV are still in infancy stage and human safety is yet to be tested) Pre existing immunity (for Ad and vaccinia virus it is serious while for zoonotic vaccine vectors it is not problematic) Vector’s genomic capacity for a transgene insert (exogenous DNA can be in the range of 1 kb to 35kb depending on the vector) 35