I feel only the increased antibody titre will not signify the efficacy of the vaccine. If the survival of fish is positively co-relating with the titre it can defiantly be a sufficient and good measure of efficiency. And it differs with different pathogens. Some may show good antibody titre but no co-relation with survival and vice versa.

Well, you have the survival data which are remarkable, so your association of antibody rise only in the vaccinated fish with the efficacy of the vaccine is very strong and could make you rely on the former as a good marker.

Importantly, are the control fish vaccinated with "empty" L. Lactis vectors?

This is important to rule out a defense mechanism towards the vector and not to the vibrios' proteins.

In the end, protection is the only measure of vaccine efficiency. So the question is "Is antibody titre to the recombinant protein you inserted into L. lactis a useful predictor of efficacy?" Really elevated antibody titres say "yes the fish was immunized and the immunization is roughly focused towards humoral immunity. You don't know which class of antibodies are being made and whether cell mediated immunity is important in protection. At least some Vibrios are able to become intacellular so CMI may be important. If CMI is important high antibody titre may be a negative predictor.

Have you looked at persistence of the L. lactis strains on your fish? If it is somehow persistent it changes the whole dynamic of immunization.

Also, I am curious about the number of fish in your vaccine and control groups. 100% protection always makes me nervous ........ Are you confident that your vibrio infection protocols in some way mimic reality = natural infection. Does the protection last long enough to be considered a vaccine response (months to years) or may it be something nonspecific effect of the probably massive numbers of vaccine bacteria you are adding. Are you adding equal numbers of lactis without recombinant protein to your "unvaccinated" group.

That said your results are very interesting and I hope your project goes far....

First, thank you all for the interesting comments.

1. Yes Pricilla, we had 3 treatments; Treatment A; fish fed with the modified Lactis vector, B; fish fed with the unmodified or "empty" vector and C; fish fed the diet without any vector at all. fish were all fed for 6 weeks, then all dip challenged (vibrio concentration 10^9 per ml for 3 hours), in 3 replicates of 100 fish per tank. ALL fish for treatments B (within 10 days) and C (within 2 days) died. Antibody titers for B and C at 12 hrs post challenge (when the highest level was detected) were similar, but very significantly lower than for A. Vibrio was re-isolated from ALL of a large number of randomly sampled dead fish kidneys and those that survived in A, although the number of cfu were significantly lower.

2. Like I said earlier (Glenn), the repeat challenge of the surviving fish was repeated only 2 months after and this time around, the antibody titer level was even higher than was recorded in the same fish during the 1st challenge and the survival rate is still at 100% although the number of fish has reduced to 40 per tank now, obviously due to culling after the last experiment, for analysis. We hope to keep them for 2 more months and then stop the experiment.

3. I was looking for opinions with regards what other analysis is thought necessary in support of the mortality data, antibody titer and other blood chemistry parameters already in progress?

Thank you.

Ah, thanks, very interesting! so you do see a difference in time to death (2 versus 10 days) in the two control groups, whereas the antibody titers were similar, perhaps suggesting there could be a cellular (non antibody) response to the vector which however is not protective? I don't know anything about fish immunology... but isn't there a cytotoxic activity of that you can measure?

Other parameters .....if you can measure them. 1) Cytokine response to infection and vaccination in fish => Th1/Th2/Treg. 2) Persistance of the lactis strain on fish 3) Make polyclonal or monoclonal antibodies to your vaccine protein to determine if they can passively protect fish. 4) If your vaccine protein is an outer membrane protein = determine if the protein can block adhesion/ invasion of vibrio into fish culture cells (if you have appropriate fish cell culture cells)

Dear Mohammed,

It sounds interesting. I think it is better to check a series of immunity parameters in treated group (vaccinated fish) and then compared to untreated (control) to elucidate which factor(s) may cause the survival of your tested fish. Some of them are listed below:

Lysozyme activity

Complement activity

Leucocyte (WBC) counts,

Oxidative radical production (NBT activity),

Total plasma protein (TP),

Total immunoglobulin (TI) levels,

phagocytic activity (PA)

and, etc.

Your challenge concentration is very high and vibriosis rarely provokes 100% mortality. When we do Vibrio challenge in sea bass, we usually use 10^6 up to 2.10^7 cfu/ml maximum depending on water temperatures and fish weights in order to get a mortality of 30-50%. Anyhow, your results are very good especially for an oral vaccine but it may be worthwhile to try lower concentrations of Vibrio closer to those found in fish farms faced with the disease. You can measure a lot of immunological parameters but in substance, the bacterial challenge is the final and most important result when studying vaccine efficiency.

OK, biomedical sciences are not my cup of tea, but I would like to make some remarks.

Firstly, you are not informing us how many fish you have in each arm. Additionally, 100% mortality could be interesting, but using survival models like life tables (Kapplan-Meier or actuarial life tables) or survival analysis (Poisson or Cox regression) could be cool to add.

Yes, I am agree with Hedley! You can use Survival Test (Cox Regression or Kapplan-Meier). Please let us which species did you use during your experiment?

I am not a specialist of fish pathogens, so I apologize for my very naive questions/suggestions . Have you looked at reduction of your pathogen titer after challenge ? I do not know if it possible in your system to have non sterilizing protection. I would suggest, if you have not already done it, to include a control group vaccinated with the empty vector, to evaluate the role of the L.Lactis backbone in protection. Again, I do not know how it works in fish, but in mammalian we usually challenge at distance from the peak of the Abs. So may be you could test a single challenge dose but at different times post-vaccination (perhaps combined with a vaccine dose-effect). You have observed increased titers after the 2nd challenge but did you look just before this 2nd challenge ? Your titer may have decreased between the 1st and the 2nd challenge, which could explain this nice booster effect

Again thank you for the interesting comments which this discussion thread has generated.

1. Pedran, most of those assays have been taken into consideration, the few you added would be considered. Thank you.

2. Morgane, based on the literature and our own previous work 10^6 cfu/mL is only effective when challenged IP. when you dip challenge, that concentration does not work even for non-vaccinated fish. Moreover, or fish species used is tiger grouper and they weighed less than 10 g. We came up with the 10^9 after preliminary trials. Anyway, your observations are noted

3. Hedley, you may need to read my earlier posts. I did mention that for each treatment, we had 3 replications of 100 fish each, making it 300 fish per treatment. What I meant by 100% mortality was that ALL the 300 fish for treatments B and C (defined earlier) died, whereas 100% survival meant ALL the 300 fish for treatment A survived. Regardless of the method of analysis u are suggesting, I am afraid it would still come to 100% dead or 100% survival; in other words, we would end up with a 1 or a 0.

4. Veronique, very inciting and thought-provoking "naive questions" there and I like them. My next question to all contributors; What time interval would anyone consider it appropriate to challenge vaccinated fish, after oral administration of vaccines? This is because there are conflicting information in the literature, where challenge immediately after vaccination is said to give "wrong signal" of protection.

Thanks again

Hi Paiko, I think you might as well need to consider further trials that gauge interractions of practical conditions with vaccinal efficacy. In other words how robust will the protection be in fish already disturbed by stressful practical conditions? This is key as we can expect that day to day on farm growing conditions that stress animals do interfere with drugs or vaccinal efficacy. In this regard, I suggest that you set up treatments that include fish previously challenged with toxins (could be feed born, or chemical toxicants), physiological or nutritional stress. Stress models you may test would definitely abound in your subject specific literature. Going this far could hint what are likely outcomes in the field when your product (hope it becomes one) gets to the market space. In poultry that I am well conversant with, we noticed that when birds are feeding diets ladden with mycotoxins or challenged by thermal conditions, efficacy of vaccines like newcastle or Gomboro vaccines are drastically reduced. Infact the vaccines often breakdown. Same story for antibiotic drugs- we need to apply more for the same effect. Hope this helps.

Hi Ola, thank you for the interesting insight. We would consider these other trials suggested as time goes on but first, our interest is to be sure that what we are observing in these initial trials clear the laboratory-scale questions, as much as possible. The field trials taking the factors you mentioned will be our next interest.

S. B. Fontal, I am not sure I understand your question very well. However, all I can say is that the relatively few (significantly lower than in both controls) pathogens we isolated from the vaccinated fish at the very beginning is what we considered to mean that though the vaccinated fish were still infected, the number of pathogens that gained entry were too few to lead to any deleterious effect to the fish. After a few days when we checked again, the pathogens had completely disappeared, which we interpreted to mean that the immune system of the vaccinated fish was effective in dealing with them. Since we did not expose the vaccinated fish to any new other groups of fish at the time we noticed the few infections, I am not sure I am competent to answer that question at this time.

By and large, I think vaccination does not necessarily prevent pathogens from infecting fish (or any other animal for that matter), but rather it "teaches" the immune system of the fish to be more alert to its responsibility, hence making it more effective in preventive mass infection of those pathogens, to reduce the chances of diseases occurring. As consumers, we do encounter microbes (good and bad) in products, the difference between a "healthy" fish and a "diseased" one is certainly, partly in the number of pathogens and the serious damage they inflict on different tissues. Since the bad ones are usually few in the healthy fish, they could easily be dealt with when we prepare the product for consumption, I think.

I hope this helps.

Thank you

Dear Mohammed, when challenged with pathogens, an increased antibody titre occurs only if the agent had induced a B cell memory response. Some antigens or pathogens are known to induce T cell memory response more than B cell memory. In such cases increased antibody titres may not be evident however, there may be an increased levels of cytokines that are involved in T cell memory like IFN gamma and IL-2.

Dear Mohamed

I think that this working is very interessent : I have no experience with vaccine of fish but in analogy I can suppose that :

- you can control the seroconversion in time ( 15 days, one month, 6 months.... you can have an idea on the cinetic of production of antibodies of your vaccine, you can appreciate the stimulation, and also you can mesure the cellular immunity if there are one in this case.

Efficacity can be measurated by the production of antibodies in the vaccinatad animal, but the real potency of your vaccine can be tested by challenge with wild strain.

You can use the challenge if you had good candidate. After 6 months or later post vaccination you can determine the valididity in time of the protection against a wild strain. When 80 % of non vaccinated dead by the infection of the wild type.

good continuation Soory for my English

Dear Mohammed,

I think the duration of protection is one of the most important parameteres that you should take into consideration. Therefore, is is better to keep your vaccinated fish for a long time (i.e. 6 to 12 months) to investigate the durability of produced vaccine.

In addition, you should check your vaccinated fish for control them for LATENT of CAREER phase.

Good luck.

Braimi and Pedram,

thank you both for your invaluable contributions.

However, I need to state clearly that the vacines proposed are for marine fish whose production cycle (from fingerlings to marketable size) does not exceed 8 months. The whole idea is that the vaccination targets to give protection within the production cycle. I will keep your suggestions in mind.

Furthermore, I wanted Nagendrakumar (or any other person) to explain (if they could, please) how we can experimentally differentiate between the pathogens we challenge inducing B or T memory cells response. The explanation offered earlier caught my attention and I was thinking it would be worth-while to experiment this.

Once again, thanks to all contributors in no small measure

Hi,

In answer to your main question antibody titre alone is not indicative in fish that there is a protective immune response so the quick answer is that measuring antibody titre is not sufficient by itself as some aquatic microbial pathogens evoke a cellular immune response and the humoral (circulating antibodies) values might be high when measured post-antigen exposure, but will not give a protective response when the fish are then exposed to the pathogen after vaccination. You may have already said this in a previous post but it would be good to know what adjuvant (if any) were used with your vaccine? Did you use an adjuvant only group to compare the antibody titres between the groups? Your results do seem to suggest that you have a protective response from the "vaccine" administered but a control group is required (I think that you have included that already but just asking again), which should be a control group for the vaccine as well as the pathogen and the adjuvant (if you added any) - if no adjuvant added then the control group without pathogen would do. I agree with the comment by Pedram that you do need to see the proportion of fish at the end of the trial that still have the bacteria. Happy to discuss this further if it is helpful.

Good luck!

Dear Mohammed,

Some questions formed in my mind about your assay.

1)Which method you employed for Antibody titration?

2) Which antibody was measured in your assay? (IgM or IgG)

3) which serology method was applied?

It would be appreciated if you answer my questions for more discussion.

Maybe, we can share our experiences in this matter.

Thanks and regards

Hello Dr. Paiko

At the outset, I congratulate you for developing a good Recombinant Vector Vaccine.

One of the most important criteria of an ideal vaccine is protective immunity (other criteria being SAFETY (the vaccine should not cause the disease!) and IMMUNOGENICITY (the vaccine should be able to induce a vibrant immune response).

Thus an ideal vaccine should be able to protect the host against the infection. Hence, I think the most important efficacy test is the disease resistance test (Challenge test) which you have done 2 months after vaccination. Since the culture period of your fish is about 8 months, I would recommend that you do challenge test 4 and 8 months after vaccination. Calculate RPS (Relative Percent Survival) at these time points. If they are again 100, your vaccine is an excellent one. But if it is substantially lower than that at 2 months point (which is 100), say 60 and 40 respectively, then you have to think in terms of giving one or two boosters to increase the RPS values to closer to 100.

All other assays like antibody titre, IL2 etc are all indicators of possible protection. Sometimes, there may not be even positive correlation between the level of some of these parameters and the degree of protection (for example, if the increased antibodies produced were not against the protective/critical epitopes of the pathogen, then there may not be corresponding increase in degree of protection!)

All the best, RD Michael

I think that memory cell that responsible for antibody response during the second exposure to the same pathogen was a result of antigen introduced. This may be one quality of the vaccine in that its antigen is responsible for specific memory cell production. In that case, since memory cell was sensitized at the second time exposure, this memory cell production reflects efficacy of vaccine. Therefore, this memory cell production may reflect vaccine efficacy.