“What is next in PRRS vaccination: Pursuit of broad heterologous protection”

A presentation prepared for the 24th International Pig Veterinary Society Congress and 8th European Symposium of Porcine Health Management at the Royal Dublin Society (RDS) Dublin, Ireland 8th June 2016.

Porcine reproductive and respiratory Syndrome Virus (PRRSV) is the most economically significant infectious disease of swine worldwide. Due to the great impact of PRRS in many key swine producing areas of Asia, Europe and the Americas, the epidemiological situation created by this infection has become very complex. Perhaps an accurate picture of the current worldwide situation of PRRSV had been already anticipated in a conference offered in 2008 by Dr. Jeffrey Zimmerman ( Iowa State University) during that year’s London Swine Conference ( London, Ontario, Canada) where it was said : “This period ( last decade) has been marked by 1) A growing recognition of the high cost of PRRS to swine producers; 2) Continued producer frustration with the poor control of PRRS; 3) Heightened interest in regional elimination of PRRSV, but reluctance to proceed without more reliable methods of achieving the objective; 4) Reports (and “counter reports”) of newly emerging, highly virulent, PRRSV isolates; and 5) Innovation in the application of diagnostics to surveillance”.

Certainly the challenge in the PRRSV arena resides on the development of the tools that are necessary to warrant a technologically sustainable eradication of PRRSV. The research community can currently be described as “tri-fold split” in relation to which priorities should be assigned in order to achieve such technological sustainability: 1) Many would emphasize the idea of immediate eradication with strict management of biosecurity, with very meager and frustrating experiences as in many areas of dense endemicity and swine intensive production it becomes almost impossible to maintain large herds free of PRRSV infection 2) others are cautious about that previous approach and bring the fact that no major endemic infectious disease, human or animal, has been eradicated without the help of an efficient tool such an effective viral vaccine. Such tool, as needed for the case of PRRSV, is not here yet, but active research towards such goal is ongoing in America, Europe and Asia. We plan to update this IPVS audience on the efforts along that line. 3) Finally an additional group of researchers seem to be ready to argue that characterization of genetic control of resistance to disease and recent advances in transgenic pig studies may provide the solution to the PRRSV situation. Such view will be also presented by other speakers at this meeting today.

A major research goal of our laboratories at the University of Nebraska is the development of a new generation of PRRSV differential marker vaccines that would confer broad protection. Such goal is based on a major premise: the conviction that the use of vaccines will always be a cost-efficient method and the preferred approach to control PRRSV infections. As early as June 2007, a nationwide colloquium held by US PRRSV experts at the University of Illinois (Urbana-Champaign) (7) came to the conclusion that a new generation vaccine for PRRSV may require at least 10 years to reach the market ( an accurate time prediction still valid today(2016) !), and that several technical approaches may be followed to develop such novel vaccine. However, the expert group concluded that the live, replicating type of vaccine seems generally to be the most favored, based on the more robust immunity that can develop in the pig after the application of this type of vaccine. This presentation will summarize the different avenues being explored for possibly improving the broadness of the coverage provided by current PRRSV vaccines. To improve the current vaccines it is essential to understand the basis of protective immunity generated by wt PRRSV upon natural infection.

What is currently known about PRRSV protective immunity

Our current knowledge on the basic mechanisms for PRRSV protective immunity is fragmentary. A significant degree of genetic diversity amongst the PRRSV strains circulating in the field exists. Likewise, a clear definition of what is meant by effective heterologous protection amongst PRRSV strains is still lacking. We have studied the resolution of persistence of wt PRRSV in convalescent animals. Contrary to other known examples of persistent RNA viruses, the persistence established by PRRSV is finite and seems to involve a low level of productive infection which progressively declines ( i.e smoldering infection) until complete viral clearance takes place. During viral persistence, extensive modulation of the innate and acquired immune response takes place, although finally a firm convalescent immunity gets established and PRRSV is eliminated from the infected pig, although this cure sometimes takes up to 5+ months(1).

Using reverse genetics we have investigated the major modulating effects exerted by PRRSV on immunity and the main viral structures involved in that process.

A major immediate effect on the innate response upon PRRSV infection is a severe inhibition of 2 pro-inflammatory cytokines: IFN type I and tumor necrosis factor alpha. Such subversion of the pig’s innate immune response seems to be primarily (although not exclusively) mediated by the two subunits of the nonstructural protein 1 (PRRSV NSP1 alpha and beta)(3, 17).

Regarding acquired immunity, perhaps the most compelling example of PRRSV modulation of the immunity is given by the aberrant timing of appearance of PRRSV-neutralizing antibodies. The PRRSV-neutralizing antibodies, that we have shown are a major correlate of protection, are produced by the pig very late in the infection process (at around 6 weeks p.i.). (11)The mechanisms of modulation of the acquired protective immune response by PRRSV may involve different strategies of immune evasion, including the display of decoy epitopes in the proximity of neutralizing epitope of the major envelope glycoprotein GP5(15), as well as glycan shielding of at least two envelope glycoproteins, GP5(2) and GP3(21).

To complete the puzzle, there still remains to ascertain the contribution towards protective immunity of other components of PRRSV. However, given the significant strain diversity of PRRSV, the major challenge for improvement of current vaccines consists of finding the basis for broadening their protective response,

What is being done to improve PRRSV vaccines, New knowledge on PRRSV biology, new PRRSV technology and PRRSV effective vaccination... Are we there yet?

It is well accepted by now that the establishment of an efficacious immunologic memory specific against PRRSV will require mechanisms of antigen presentation that would mimic those used by the wild-type live PRRSV itself. In that respect the main avenues followed by different laboratories seeking the development of new generation vaccines X PRRSV are varied, although centered around two principal, alternative principles: 1) use of viral vectors that would replicate in vivo and would present PRRSV antigens to the immune system just like live PRRSV does. This progressive approach still awaits for more clear information on the complete set of immunogenic structural subunits that should be required for the establishment of an effective protective immunity, 2) the use of reverse genetic technology ( infectious clones) for the improvement of the current MLV vaccines or the de novo development of live vaccines that would be rationally attenuated and endowed with a capacity to differentiate infected from vaccinated animals ( i.e. fulfilling DIVA principles).

Above and beyond of the method(s) that eventually prove(s) to be successful in reaching effective homologous protection, a formidable additional challenge in all cases is represented by the need of defining the number of valencies or specificities that should be used in the formulation of the novel vaccines in order to ensure broad protection against PRRSV infection. The major research investment in the area of PRRSV in the US has certainly been on vaccine research and development. Although the current modified live vaccines may confer a solid protection when the infection or challenge of vaccinated animals takes place with a PRRSV strain of antigenic make-up similar to the vaccine strain, the protection against heterologous strains (i.e.: more distant from the vaccine) remains less than desirable. On the other hand, authentic evidence for any measurable level of protection obtained with inactivated (killed) vaccines remains to be obtained. Many experts also express doubts that PRRSV control and eventual elimination could be achieved without broadly protective vaccines that reduce shedding and transmission. Although more than 25 years have elapsed since the discovery of PRRSV, much remains unknown, as mentioned above, about the immunology of this virus. In particular, the exact nature of the protective immune response remains unclear and the goal of broadly protective vaccines for PRRS has not been achieved, due in great part to the highly variable nature of PRRSV, as well as the significant modulating effect that PRRSV infection seems to inflict on the host’s immune response. Development of adaptive immune responses after infection of naive pigs with PRRSV or vaccination is known to be anomalous, with IFN gamma-secreting cells appearing late and evolving erratically during the first weeks after infection(11, 12). Appearance of neutralizing antibodies is also delayed. Neutralizing antibodies may protect against infection if present in the body in sufficient quantities before infection, but they are not effective at clearing PRRSV during natural infections. PRRSV is able to modulate innate responses, probably through the regulation of IFN-alpha, other pro-inflammatory cytokines, and perhaps, IL-10. While the mechanisms of protective immunity against PRRSV may not be fully elucidated, research from our own laboratory as well as others clearly indicate that PRRSV-neutralizing antibodies are important contributors to PRRSV protective immunity(10, 14). However, while it is clear that antibody may confer complete immunity to PRRSV, experiments indicate that other mechanisms, such as innate and/or cell mediated immunity, may also be protective (25). Further complicating immune protection against PRRSV is the pronounced diversity of this virus (13). PRRSV exists as two major genotypes, European (Type 1) and North American (Type 2). There is only limited immunologic cross-protection between isolates with these genotypes. Moreover, considerable variation exists between field isolates of each of these genotypes indicating continuing divergence of viral genomic sequences. The genetic diversity within each genotype is so elevated that allows a vaccinated animal to be re-infected by a different strain of the same genotype, a circumstance which further confounds our understanding of the host -PRRSV interaction. Sequence divergence has been shown to occur on serial passage between pigs, within persistently infected pigs and in pigs from infected farms. Furthermore, it is possible that different PRRSV strains are able to influence the immune system in different ways, which adds significantly to the confusion prevailing in this field(6).

Control of PRRSV has proven difficult, even with vaccination and protection against PRRSV infection remains a matter of primary importance for swine producers. Current PRRSV vaccines include 2 main types of products: modified live and inactivated virus adjuvanted vaccines, with the occasional use (mainly in the US) of inactivated autogenous vaccines made from indigenous field isolates. In June 2007, a meeting was held at the University of Illinois, College of Veterinary Medicine to discuss the state of current knowledge about PRRS vaccination(7). The meeting was attended by invited experts in PRRS, virology, immunology and vaccinology, as well as industry and major swine veterinarians. Major conclusions of this colloquium were that successful vaccination against PRRSV can be achieved and improved, with current MLV vaccines as the gold standard by which improvement is defined. The group of experts, however, advised that a final product would feasibly not available in at least 10 years and that three major technical challenges or knowledge gaps need to be overcome before such successful development of an effective new vaccine takes place. These are: 1) to identify structural components of PRRSV and host mechanisms involved in PRRSV protective immunity, 2) to understand the mechanisms involved in PRRSV attenuation in order to reduce virulence and/or increase immune responses of the vaccine strains and 3) to improve the meager protective efficacy of current vaccines against heterologous PRRSV strains, mainly by ascertaining what defines a heterologous strain in terms of protective immunity. Multiple evidences indicate that the best protection that can be obtained when vaccinating pigs is through the use of replicating, attenuated live vaccines(25). Inactivated vaccines, as we said above, appear to be ineffective(25). In addition, some immunogenic PRRSV structural subunits are being tested in different laboratories worldwide. Attention focuses specifically on the GP5/M hetero-dimer cloned in vectored vaccines. The GP5/M subunit vaccines produced are so far able to confer a relative level of protection, insufficiently protective and lower to that attained with live vaccines. This indicates that more needs to be known about the immunogenic role of other PRRSV structural components. Work in our laboratories at the University of Nebraska (4) points towards the important notion that GP2 and GP4, two minor glycoproteins located on the surface envelope of PRRSV and that have been the target of little investigation so far, are the viral components that interact with the virus receptor of the host cell Such role would make GP2 and GP4 central to the induction of a protective/neutralizing host response. These subunits, therefore should be included in the list of immunogenic components of PRRSV that would contribute significantly to protective immunity against PRRSV infection. It appears that there is a significant cell-mediated immune protection that can only be conferred, so far, by live vaccines, thus the inclination expressed by the expert groups at the Illinois symposium, towards the live vaccines, which in some cases have been already designed, through reverse genetics, as differential (DIVA) marker vaccines. Independently of the platform and technology to be used for the design of new generation PRRSV vaccines, it is clear that a major challenge yet to be overcome in PRRSV vaccinology should be that of circumventing strain diversity so to obtain a widely protective immunogen as stated in the conclusions of the Illinois PRRSV Vaccine Colloquium. Modified live PRRSV vaccines can confer homologous protective immunity that is considered to be close to or completely sterilizing immunity. On the other hand, however, the extent and duration of protection against heterologous strains may be variable and dependent on antigenic relatedness of the virus strain used for inoculation and challenge. Percentages of heterologous protection attained by MLV vaccines in pregnant sows have been described to vary widely: ranging between 52 % and 85.9 %. The wide range of heterologous protection empirically obtained by commercial vaccines dramatically exemplifies our current difficulties at precisely defining a heterologous level of protection in PRRSV.

Can we group the PRRSV strains (and so define valencies to be contained in vaccine) based on their antigenic rather than their genetic phenotype?

Phylogenetic analysis of hypervariable structural genes of PRRSV such as GP5 has been used extensively to group and study relationships between strains. Besides the GP5-based classification, no other antigenic grouping of the constellation of strains of PRRSV has been attempted until recently. Research now being conducted at the universities of Nebraska and Complutense of Madrid involves attempts to use cross-neutralization to systematically group PRRSV isolates by some important phenotypic trait such as the strain’s antigenic make up. The notion that GP5 sequence alone is not a good predictor of the protective effectiveness of a vaccine strain and the realization that other possibly protective genes different than GP5 may induce neutralizing antibodies emphasize the need for better knowledge of other viral genes important for cross protection and broad antigenic coverage by vaccines. However, the level of knowledge at the level of the entire genome of PRRSV had been very scarce, with a limited number of strain genome-length sequenced until a few years ago, and many of these closely related sequences and/or lacking phenotypic data.

How to widen the cross protective effect of the current vaccines?

Different research teams are pursuing the goal of expansion of protective coverage of PRRSV vaccines following independent separate approaches. Overall, we can list at least three main lines of work that are directed at broadening the antigenic coverage of the PRRSV live vaccines:

In one case, the emphasis is being placed primarily in discovery, characterization and establishment of strategies to develop immunogens oriented at inducing broadly neutralizing antibodies that would be capable of inactivating antigenically diverse strains of PRRSV (16). The notion in place in this case is that the broadly neutralizing antibodies would be directed to conserved epitopes domains such as the contact residues that interact with the most significant cellular receptor for PRRSV (CD163), based on the CD163 interaction between the glycoproteins trimer as proposed by Das et al. (5), although recent solid evidence for the occurrence of broadly neutralizing antibodies may also occur in the matrix envelope of PRRSV(20)

Two other innovative approaches recently used to explore the construction of broadly protective PRRSV immunogens that would circumvent the heterologous variability of strains are based on bioinformatics analysis of the PRRSV genetic variability. Both of these approaches seek the design of immunogens that respond to the notion of centralized antigens that provide broad protection as originally described for human immuno-deficiency virus (8, 9). In one case ( Virginia Tech University) the application of this technology to the PRRSV model has centered in using one or more structural glycoproteins designed as antigenic mosaics (19, 23, 24). Alternatively, another recent application of the centralized mosaic principle to on the design of a PRRSV live replicating immunogen ( University of Nebraska) has been based on transferring over the concept of a single structural centralized antigen to the entire PRRSV genome, which can be virtually be considered as multiple centralized antigens of PRRSV( i.e including all structural and all nonstructural genes) coded for throughout the entire PRRSV genome(22).

XJ Meng’ s lab (Virginia Tech campus) has extensively studied the adoption of the mosaic vaccine concept to the PRRSV model by means of molecular breeding through DNA shuffling, a process that would essentially mimic natural recombination but that forces it to happen at a much accelerated speed in a cell culture plate rather than in vivo. These authors developed initially a mosaic of PRRSV GP3 and PRRSV GP4 and M, while more recently they reported a mosaic containing all the structural genes shuffled for 6 heterologous strains cloned inside a backbone of a commercial vaccine, thus obtaining a replicating construct that they tested to be used as an experimental vaccine. While single shuffled GP DNA vaccine was useful to show some moderate cross protection as expressed by some sober cross-neutralizing titers, when the vaccine was formulated as a mosaic composed of all the structural genes of six donor heterologous strains, then exhibited some level of cross protection against heterologous strains challenge, although just a partial one.

On the other hand, Vu et al (University of Nebraska) used a set of 60 non-redundant, full-genome sequences of type-II PRRSV to generate the consensus genome (PRRSV-CON) by aligning the 60 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. The resulting PRRSVCON has the highest degree of sequence identity to the PRRSV field-isolates when compared to any current PRRS vaccine strains, both at the full-genome level and the individual gene level. These authors then synthesized an entire PRRSV-CON genome and pursued classical reverse genetics to generate PRRSV-CON cDNA clone which proved to be fully infectious and virulent when the PRRSV was recovered by classical reverse genetics. Cross protection trials were conducted using groups of recently weaned pigs assessing, viremia, average weight gain, viral load in tissues, and lung pathology against distant (widely heterologous) challenge strains. Remarkably, primary infection of pigs with PRRSV-CON virus conferred significantly broader protection than the prototype PRRSV strain FL12 when tested upon subsequent independent challenge trials with two unrelated widely heterologous PRRSV strains.

Summary/ Conclusions/what is next?

Bio-informatics analysis of PRRSV genomes may provide the clue for broadening the antigenic coverage of PRRSV vaccines and immunogens. DNA shuffling offers an opportunity for rational design of PRRSV MLV vaccines that possibly confer heterologous protection at the level of single structural genes or in the format of mosaics of multiple shuffled structural genes. In addition, shuffled PRRSV chimeric antigens, when targeted through DC-SIGN directly to DCs, elicited antigen-specific T cell immunity in pigs (18).

The synthetic PRRSV-CON virus can serve as the parental strain for the development of a novel PRRS live vaccine with broader cross-protection. The PRRSV-CON virus confers exceptional cross-protection against divergent PRRSV strains thus serving, as the current gold standard for PRRSV cross protection between heterologous strains. The PRRSV-CON virus can serve, upon adequate and complete attenuation, as a potential seed strain for the formulation of a broadly protective live attenuated PRRS vaccine. In addition, the occurrence of an exceptionally board protection like the PRRSV-CON virus provides an extraordinary reference tool to ascertain the mechanisms and correlates implicit in heterologous protection against divergent PRRSV strains.

Presented at the 24th International Pig Veterinary Society Congress. For information on the next edition, click here.

“What is next in PRRS vaccination: Pursuit of broad heterologous protection”

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