Piggy-backing flu on smallpox vaccine

There is as yet no pandemic bird flu vaccine but there are a lot of potential vaccines. The recent fiasco involving Baxter International (here, here) involved one in development. There are many more. They employ old and new technologies and are in various stages, a few in early clinical trials. Many more are in the pre-clinical (animal or test tube) phase, although they are frequently reported in the news because the company developing it wants to attract support or publicity. I often don't pay attention to announcements of "breakthroughs" that are successful in mice. Many vaccines work in mice but not in humans or work in mice and can't be used in humans because of side effects. This weekend we are brought news of yet another of these. It was also announced at a press conference, but no company's name was connected with it, the paper is just published in a peer reviewed journal, and no financial conflicts or commercial research support is listed. The money comes from NIH and the team includes highly experienced and respected flu scientists. The news reports of the science were interesting enough I decided to invest a few hours in reading the paper, entitled "Vaccinia Virus-Based Multivalent H5N1 Avian Influenza Vaccines Adjuvanted with IL-15 Confer Sterile Cross-Clade Protection in Mice" (Poon et al., The Journal of Immunology, 2009, 182: 3063-3071 [subscription required]). Here's the description from Reuters:

Scientists in Hong Kong and the United States have developed an experimental H5N1 bird flu vaccine for people by piggy-backing it on the well-tested and highly successful smallpox vaccine.

Initial tests on mice showed the vaccine to be highly effective, they told a news conference in Hong Kong on Sunday.

[snip]

Smallpox was eradicated worldwide in 1979 and the experts are hoping that their novel H5N1 vaccine can ride on the various advantages of the smallpox vaccine.

The smallpox vaccine is very cheap, has a long shelf-life of several years and does not require highly sophisticated laboratories, making it easier for poorer countries to produce.

"It is very stable and you can pack them off to developing countries and use them. They require refrigeration but it is less critical than other vaccines," Peiris said.

"Smallpox production capacity has gone down but many countries have the technology and the expertise to do it, and if necessary, it can be very quickly scaled up."

"But for other strategies (of producing H5N1 vaccines), it is not possible to rapidly set up manufacturing plants all over the world as they require very specialised plants." (Tan Ee Lyn, Reuters)

This gives the gist, but as usual there is a bit more to it. Vaccinia virus is the virus of cowpox, a much less serious disease than smallpox. If you can be made immune to cowpox you will also be made immune to smallpox, something demonstrated by Edward Jenner in 1796. The word vaccination comes from the Latin word for cow (vacca), as in cowpox. When we are intentionally infected with vaccinia virus (via a scratch on our upper arms), the infected cells make copies of the virus and the proteins on the virus induce the immunity. For most people with healthy immune systems, vaccinia infection is mild and long lasting immunity to smallpox virus results. Because humans are the only reservoir for smallpox, it was possible to eradicate the disease by systematic vaccination campaigns. The last death from smallpox occurred in 1978. Ironically, it was a result of a laboratory accident, and more ironically, still occurred on September 11 of that year.

What the newly announced mouse vaccine did was insert into the vaccinia virus the genes for five proteins made by H5N1, the bird flu virus. These genes are the ones that make the hemagglutinin (H5), neuraminidase (N1) proteins, the matrix protein, M1, the ion-channel protein M2 and the nucleoprotein, NP. Most of these proteins, including H5, are not very immunogenic in humans, i.e., they don't produce strong antibody reactions, so additional materials, called adjuvants, are frequently added to vaccines to boost the immune response. A standard adjuvant is one made of an aluminum compound, but now there are others, some of them proprietary so we don't know much about them. These extra materials also increase the likelihood of side effects. The new vaccine used a different kind of adjuvant, a naturally produced immune system modulator called a cytokine. Along with the genes for the viral proteins they also inserted the gene for the human cytokine, interleukin-15 (IL-15). IL-15 stimulates various components of the immune system, so the idea was to use this innate biological mechanism as an adjuvant. On each of these genes a sequences used by the vaccinia virus to start and stop the production of proteins from genes was added and all of them lined up end to end and put into the vaccinia virus. Now when the virus infected the mouse cells, they produced not only vaccinia proteins but also the added proteins from the H5N1 and IL-15.

That's the basic idea, but there are a lot of i's to dot and t's to cross when you do science, and most of the 9 pages of this paper are taken up with those matters. For example, the research team made vaccinia vectors that had a the full-length IL-15 gene with a mutation that made inactive IL-15 so they could see what the effect of the adjuvant was. They also had a vaccinia virus with only IL-15 added, so they could see the effect of the viral proteins. They checked what was going on in many different ways. For example, they measured the level of certain antibodies to see what the vaccinia virus was producing and they checked to see if the antibodies being produced could react to more than the specific virus whose genes were used. The ability to react more broadly than specific strain could mean that they had a vaccine that could be used before a pandemic materialized. At the moment we have to wait for the pandemic to start because we can only make vaccines against very specific strains. They also challenged mice with H5N1 to see how well the vaccine protected them and how quickly the protection took hold.

Finally they tried the same tricks (although only using two of the viral proteins, HA and NA) with a replication-deficient vaccinia strain called MVA (Modified Vaccinia Virus Ankara). MVA replicates very poorly in mammalian cells, but still makes large amounts protein. It is therefore useful for people with HIV or other immune defects who might be endangered by a live virus vaccinia vaccine that replicated well in human cells. Generalized vaccinia is a life threatening disease. With tens of millions of HIV positive people, an MVA-based vaccine could still be used. Unfortunately, these viruses are still grown in eggs, a serious problem because there aren't enough eggs in normal times to make sufficient vaccine if a pandemic strikes and the virus itself could exacerbate the problem by wiping out poultry. Presumably this problem can be solved if the overall strategy is successful.

And if you are a mouse, you have cause for optimism. Viral challenges that were 100% lethal in unimmunized mice were 100% protective in immunized ones, whether or not IL-15 was involved. Moreover the antibody response was long lasting and appeared much sooner (9 days versus 28 days) compared to the only FDA approved vaccine now being stockpiled by the US government made by Sanofi-Aventis. The Sanofi vaccine is an inactivated (i.e., not live virus) detergent extracted vaccine with added chemical adjuvant. It's active ingredients are sub-fractions of viral proteins. In addition, the new vaccine's neutralizing antibody production held across several H5N1 variants, but not all.

This is a new kind of bird flu vaccine, but the science isn't new. Using vaccinia virus as a vector has been on the agenda for a long time and it has been accomplished frequently in the laboratory. That much I knew. But in a quick literature search I was surprised to find that protecting mice with replication-deficient MVA vectors for flu virus is also old, going back at least 15 years to a paper by Sutter et al. ("A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus,", Vaccine. 1994 Aug;12(11):1032-40; not cited in the Poon et al. paper).

This paper adds significantly to that work, however. Here's some of the authors' discussion on that point:

Our vaccine development strategy involved three elements, namely the selection of a live delivery vector, the incorporation of a repertoire of antigenic targets to achieve broad cross protection, and the incorporation of a molecular adjuvant to enhance the breadth and durability of vaccine-induced immune responses. In designing our multivalent H5N1 influenza vaccine candidates, we opted to use a live viral vector vaccinia virus for a number of reasons . . . Vaccinia virus has a proven record of efficacy and safety and carries the feasibility of large-scale manufacture in a relatively short time frame. Vaccinia recombinants are genetically stable and possess the intrinsic capacity to induce multiple arms of the immune system conferring robust and sustainable immune responses. Importantly, unlike in the case of Ad5-vectored vaccines [adenovirus vectors, another way to smuggle viral protein making genes into a live virus vaccine], pre-existing Abs [that is, pre-existing immunity to the viral vector itself] to vaccinia virus in the general population that could impede vaccine efficacy are likely to be less of a problem, because smallpox vaccinations ended in the early 1970s globally. In addition, vaccinia recombinants expressing individual influenza genes from various subtypes such as the hemagglutinin gene, nuclear protein gene, or matrix M1 gene have been shown to protect animals from a subsequent lethal challenge of virulent influenza viruses. We selected the replication-competent, FDA-licensed smallpox Dryvax vaccine (Wyeth strain) as the backbone for integrating H5N1 avian influenza viral genes, because >1 billion people have been vaccinated with the Dryvax vaccine during the smallpox eradication campaigns four to five decades ago and recently the FDA has approved its production in Vero cell substrates, thus enabling a rapid scale-up of Dryvax-based vaccine manufacturing (see U.S. Food and Drug Administration news report for September 1, 2007; www.fda.gov/bbs/topics/NEWS/2007/NEW01693.html). (Poon et al., J. of Immunology, subs. req'd, cites omitted)

What are some of the possible pitfalls? First, it is always the case that what works in mice doesn't work in humans. Second, the addition of IL-15, while improving the laboratory test measures, may also have unanticipated effects in humans. After all, turning the knob on an immune system control might easily do things that aren't so good. This vaccine also works without IL-15 adjuvant, but not as well. But if IL-15 is a problem, it still make work good enough. And while naturally immunity to vaccinia virus isn't present in people born after the 1970s, there are a lot of us older than that. Some of their results suggest that won't be a problem, but, again, we'll have to see.

Then there's the biggest wild card of all. This technology, even if highly effective (a big "if") won't be ready for widespead use for some years, probably at least three. Is the influenza virus going to be kind enough to wait around? As they say, "timing is everything."